Method for identifying or detecting genomic rearrangements in a biological sample

ABSTRACT

A method for detection, visualization and/or comparison of polynucleotide sequences of interest using specially designed sets of long and short probes that enhance resolution and simplify visualization and detection. Probe compositions useful for practicing this method and procedures for identifying useful probes and probe combinations. These methods are useful for the detection of genomic rearrangements, especially those associated with various diseases, disorders and conditions including cancer or for assessment of genomic rearrangements associated with therapy. The probe compositions may be used in kits for detection of genetic rearrangements or in companion diagnostic products or kits, such as kits for the diagnosis or assessment of predisposition to cancer such as colorectal cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 13/665,440, filed Oct. 31, 2012, which claims priority to U.S. Provisional Application No. 61/553,889, filed Oct. 31, 2011, the entire contents of which are incorporated herein by reference. On Oct. 30, 2012, International Application PCT/IB/2012/002423 was also filed with the same title, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to high-resolution, precise method for detecting genomic rearrangements in vitro using specially designed combinations of polynucleotide probes. The invention concerns accurate methods of detection and diagnosis of conditions, disorders and diseases associated with rearrangement of genomic DNA.

2. Description of the Related Art

The Multigenic Paradigm of Human Diseases

Advances in genetic analysis of human diseases have provided better insights into the molecular mechanisms contributing to disease initiation and progression. Previous associations were made between particular diseases and association and/or linkage disequilibrium to single base mutations in somatic genetic sequences or with particular single nucleotide polymorphisms (“SNPs”) in genomic DNA. Newer technologies have provided evidence that larger genetic alterations and rearrangements are associated with, or can constitute major causes of diseases, disorders or conditions having a genetic origin or basis. Disease associations have now moved from a monogenic to a multigenic paradigm where a disease's origins and progression is mainly linked to more than one single genetic mutation or origin. While these new insights provide better avenues for disease detection and treatments, they also highlight the need for combinatorial genetic analysis that goes beyond detection of single mutational events or SNPs by assessing disease associations with larger genomic rearrangements. Such combinatorial genetic analysis would provide a better, more precise and accurate diagnosis of a particular condition, disorder, disease or pathology, but would also help establishing a more appropriate medical survey, more accurate therapeutic decisions and interventions, as well as help in assessing the efficacy of such therapies and interventions.

Multigenic Causes of Genetic Disease

Genetic disorders manifesting the same or similar clinical signs and consequences can arise from both single and exclusive, or combined, mutations in various genes. Such mutations can fall within either the single base alteration and/or the class of large genetic rearrangements. A few examples of such genetic disorders are Fragile X syndrome (mutations and expansions in the FMR1 gene), Ataxia Telangectasia (single base pair mutations in either intronic and exonic sequences as well as deletions and translocations of the ATM gene), Seckel syndrome (mutations as well as large rearrangements in SCKL1, SCKL2, SCKL3, PCTN and ATR), autism (mutations as well as large rearrangements in GLO1, MTF1 and SLC11A3), Spinal Muscular Atrophy (mutations, deletions, transconversions as well as cis-duplications involving the SMN1 and SMN2 genes) and myotonic dystrophy (trinucleotide/tetranucleotide expansions in DM1 and DM2).

Multigenic Causes of Cancer Predisposition

In the case of cancer predisposition, there are several examples of familial cancer predisposition syndromes for which one can nominate several causative genes for which both single base alterations and/or large rearrangements were identified.

Breast and Ovary Cancer. Causative genes: BRCA1, BRCA2, ATM . . . mutation type: higher proportion of point mutations identified so far.

Hereditary nonpolyposis colorectal cancer (Lynch syndroma). Causative genes: MSH2, MLH1, MSH6, EPCAM, . . . mutation type: equivalent proportion of point mutations has also been identified.

Multigenic Causes of Cancer Progression

Cancer progression is surely the human disease domain where the monogenic causative hypothesis was definitely ruled out since several years. First, the disease's initiation is strictly dependent of two molecular events (immortalizing and transforming) due to genetic alterations in at least two independent genes classified at either oncogene or tumor suppressor genes. Second, the disease's progression is linked to additional genetic alterations independent from the causative ones. Not only do these additional alterations play a role in cancer progression, they also were demonstrated to be the basis for appearance of resistance to therapy during treatments. Strikingly, in the list of cancer related genes, if extremely rare examples are only subject to discrete single base mutations (e.g., KRas or BRaf), the large majority is either subject to only large rearrangements (e.g., HER2, ALK . . . ) or to both single base mutations and large rearrangements (p53, c-myc, c-Met, EGFR . . . ).

The identification and characterization of multigenic conditions, disorders and diseases, including cancer, cardiovascular disease, diabetes and other heritable genetic conditions has been made difficult in part due to the imprecision of existing methods of molecular diagnosis. Molecular Combing is probably the sole approach allowing detecting all type of large genetic rearrangements (deletion, amplification, expansions, inversions, translocations . . . ) even in a complex and heterogeneous population (such as tumors).

High resolution barcodes allowing multiplex analysis of patients could help diagnostic at different level such as for patient stratification/classification and/or prognosis.

Multiplex High Resolution Barcodes for Identifying the Right Genetic Alterations as a Key Driver for Therapeutic Intervention

The Example of Myotonic Dystrophy

Myotonic Dystrophy (DM1) and Myotonic Dystrophy 2 (DM2) are two muscular dystrophies characterized by trinucleotide/tetranucleotide expansions in two different genes. If severe forms of DM1 can be clinically differentiated from DM2, milder DM1 forms are displayed extremely similar clinical signs than DM2. There is currently no cure for or treatment specific to myotonic dystrophy. However, DM1 patients exhibit Complications of the disease (heart problems, cataracts . . . ) not existing in DM2 that could can be treated but not cured. Differentiating DM1 and DM2 by the use of a multiplex assay of high resolution barcodes could thus help preventing and treating secondary effects

The Example of Hereditary Breast and Ovary Cancer

In certain countries (U.S.) detecting constitutional alterations in BRCA1/2 drives to therapeutic intervention (surgery/reconstitution). Thus, there is a clear need for an accurate diagnostic comprising all the potentially involved genes. Such a test could be made on the basis of a multiplex assay of high resolution barcodes comprising large chromosomal regions around genes known to be involved in this syndrome; BRCA1, BRCA2, ATM, ATR . . . .

DNA Damage and Response Inhibitors Example

Synthetic lethality became a strong reality for therapeutic decision to include Cancer patients in specific protocols/regimens. One of the first examples was given with the demonstration that Breast cancer patients with BRCA deficiency exhibit a higher sensitivity to PARP inhibitors, a new category of drug acting on DNA Damage and Response pathway. More recently, this was extended to other type of inhibitors in this category such as ATM inhibitors but also to more traditional anti-cancer drugs including all types of DNA polymerase and replication inhibitors.

Not only does this concept extended to other inhibitors, but it was also demonstrated that it could be extended to other types of cancers such as lung and metastatic melanoma.

Here, a multiplex high resolution barcode will allow detection of genetic alteration in genes involved in DNA damage and response that could help predicting sensitivity to this class of inhibitors. A list of such genes could include BRCA1, BRCA2, ATM, ATR, MSH2, MLH1, MSH6, EPCAM . . . .

The Lung Cancer Example

Numerous alterations involved in lung cancer could be multiplexed for a better patient classification such as:

LOH/Deletion (P53, STK11, LKB1, BRG1, KLF6);

Amplification (FGFR1, MET, EGFR, HER2 . . . );

Translocation: (ALK);

All these genetic alteration are associated to therapeutic treatments:

P53: Nutlin (low doses Actinomycin D produce similar effects)

FGFR1: Masitinib, PD173074, SU5402 TK1258 AZD4547 . . .

MET: GSK1363089, ARQ197, SGX523, XL184 . . .

EGFR: Tarceva, Erbitux, Vectibix . . .

HER2: Herceptin, Lapatinib . . .

ALK: Crizotinib

As at least 30% of NSCLCs were demonstrated to be dependent on at least one of these mutations, defining the genetic profile of the tumor could help driving therapeutic options. This could be made possible by designing multiplex assays combining high resolution barcodes covering this major genetic loci.

Localization of (Genetic) Sequences of Interest

Genetic sequence is the most fundamental information to synthesize functional protein. Alteration of genetic sequence sometimes results in loss of functional protein synthesis. In addition to alteration of genetic sequence, loss or gain of genetic sequence (copy number variation, CNV) also can be problematic for homeostasis of cellular activity. For example, loss of (functional) anti-tumor protein (p53) or gain of proto-oncogene (c-myc) results in cancer-prone cell. When such mutation happens (or exists) in germ cell, this mutation spreads whole cell in an individual who is either carrier or patient of genetic disease, or has a predisposition to cancer. The germline mutation can be heritable. These days CNV becomes more and more important to understand in the field of genetics (ref 1). However, copy number count alone is not always sufficient and it is often critical to establish the actual location of sequence elements. This is strikingly the case for e.g. balanced translocations. DNA sequencing and CNV detection methods such as array-based comparative genomic hybridization (aCGH) and quantitative PCR generally cannot detect these balanced mutations because these methods assess whether the sequence and the copy number are correct or not. FISH and its extended forms such as fiber-FISH or molecular combing can address these balanced mutations with different resolutions and precisions depending on methods.

Resolution and Precision

The use of BAC/PAC/cosmid probes on targeted regions was successfully conducted to detect large (a few kb to tens of kb) genomic rearrangements (ref 2). In these approaches, the minimum size of detectable events (e.g., the size of the deleted or amplified sequence), hereafter designated as the “resolution” of such an assay, is limited due to the large standard deviation involved in measuring probes or gaps of tens of kilobases. Indeed, in such assays the standard deviation of measurements increases with the length of the measured element. For example, a 40 kb-probe is measured with a standard deviation of ˜5 kb. Thus, if 16 measurements of a given probe are made on a slide, the precision on the size of the probe obtained as the mean value of measurements is in the order of magnitude of 2.5 kb (Considering the distribution is gaussian, and the precision is the half-width of the confidence interval, i.e. 2.sd/√n where sd=standard deviation and n=number of measurements). For a 10 kb-probe, where the standard deviation is ˜2 kb, the precision would be ˜1 kb. This illustrates the fact that shorter probes allow for better (lower) resolution.

Besides, the location of such an event (the position of the extremities of the event) may be defined with a precision (hereafter the location precision) limited by the size of the probe or gap within which it occurs: e.g. if a 40 kb probe is estimated to measure 39 kb in a sample, one can conclude that a 1 kb deletion occurred somewhere within the probe, with no further precision—thus, somewhere in a 40 kb genomic region. If the same 1 kb deletion had occurred within a 10 kb probe, the location of that deletion would be known with a better precision, as the range would be reduced to a 10 kb genomic region. Therefore, the smaller the probes and gaps, the better the location precision.

There are limits to small probes: (i) below a certain size, they become difficult to detect; (ii) they involve more complex color schemes (as there are relatively more probes); (iii) there are more distinct probes to cover a given region, and the experiments are therefore more expensive and time-consuming; (iv) most importantly, fast and reliable identification of probes, whether by a human operator or a piece of software, is easier with longer probes, as they are more readily distinguished from background. Indeed, background is mainly constituted of roughly circular fluorescent spots. When large enough, the shape of these spots allows to one to easily distinguish them from probes. However, when their size is small enough, they appear difficult to distinguish from small probes.

In operating conditions according to the invention, probes shorter than ˜3 kb are detected with a diminished efficiency. Within the 3-10 kb range, the standard deviation of measurements varies little, and there is therefore little benefit in resolution with the shorter probes within this range. Therefore, this range is usually considered to be a good compromise for probe size. However, in cases where probes are close enough (less than 10 kb gaps), smaller probes (within the 500-3000 bp range) are still useful, as they will be detected in at least a fraction of signals and the presence of the corresponding sequences may therefore be established with certainty. It was also found that detection of isolated probes longer than 12 kb (preferably longer than 14 kb) is more reliable, whether for a human operator or for automatic detection software.

Exclusion of Repeats

Eukaryotic genomic DNA contains various repetitive sequences, i.e., sequences that appear more than once (and more than statistically predicted based on their length and base content) in a normal haploid genome. Among these, some appear with very high frequency (tens of thousands to millions of copies). In human genomic DNA, the most abundant of these is the Alu family, which has ˜1,000,000 copies constituting ˜10% of the genome. In any hybridization procedure involving human genomic DNA, it is expected that probes carrying such repeats would hybridize on numerous targets, generating non-specific signal from regions throughout the genome. Other types of repetitive sequences exist, with lower frequency, and often more specific localization. The number of copies and repeat sequence length may vary widely, as well as the degree of homology. Beta-satellite sequences, for example, are present in multiple copies (hundreds to thousands), usually as tandem repeat arrays comprising hundreds of copies of the same 50-100 bp long sequence, specifically localized in a limited number of loci. Strategies to get rid of the non-specific signals depend on the type of procedure and probe. Schematically, when probes are very short sequences of DNA (oligonucleotides, typically less than 100 bp), as in aCGH procedures, the sequence of the oligonucleotides is chosen to be free of repetitive sequences, by comparison with repetitive sequences found in databases. This strategy is only practical for very short probes, as short sequences free of repetitive sequences are relatively abundant, but unpractical for longer probes, as long stretches completely devoid of repetitive elements are rare (although this has been adapted to longer FISH probes, in an approach that suffers multiple drawbacks, see below). Besides, even for short probes, it constrains the design of probes heavily and some genomic regions, rich in repetitive sequences, have lower density of coverage (and thus lower resolution of events) due to this constraint.

When probes are longer (typically PCR products or cloned DNA inserts—1 to 150 kb), in Southern Blot or in FISH procedures, non-labeled competitive DNA, enriched in repetitive elements such as Alu repeats (usually Cot-1 DNA), is added in large excess along with the labeled probe. Competition of unlabelled probes on the repetitive sequences minimizes the hybridization of labeled probes. This strategy is expensive and since the competitor DNA is not purely made of repetitive sequences, competition also occurs on the unique sequences for which the probes were designed, thus limiting the amount of competitor DNA that may be used. Therefore, the efficiency of this approach is limited.

An alternative approach for longer probes has been proposed by Knoll and collaborators (U.S. Pat. No. 7,014,997), resembling the strategy usually adopted for oligonucleotides: probes are chosen within sequence intervals devoid from repetitive elements. This strategy is based on bioinformatics analysis of the regions of interest and exclusion of known repetitive sequences by comparison with sequence databases. However, this approach has several limitations: prior knowledge of the repetitive sequences is required, which can be a problem e.g. in species where such knowledge is unavailable. More importantly, intervals longer than 2 kb devoid of repetitive sequences appear only once in 20-30 kb on average and are unevenly distributed (Considering the distribution is gaussian, and the precision is the half-width of the confidence interval, i.e. 2.sd/√n where sd=standard deviation and n=number o) so the design of probes would be highly constrained, impairing the possibility to design a high-resolution code. This would prove especially difficult in repeat-rich regions, and/or regions where pseudogenes are located next to homologous genes of interest—such low-copy repetitive sequences being also excluded with the strategy from Knoll and co (ref. 3). Since regions targeted in rearrangement tests, e.g., for diagnostics purposes, often display these features, this approach is not suitable for the design of high-resolution barcodes and especially not if such a code is to be used for diagnostics purposes. Distinctions between this approach and the invention are disclosed in more detail below.

BRIEF SUMMARY OF THE INVENTION

The present invention concerns the field of the in vitro diagnosis and detection of genetic rearrangements and is related to a method to identify or detect genetic rearrangements in a biological sample to be tested which are already known or which are new and provide markers for example of diseases as cancers or metabolic or foetal genetic diseases. The invention is characterized by using compositions containing purified or synthesized nucleic acid molecules (polynucleotides) having nucleotide sequences selected as short sequences with a length of less than 10 Kb and associated in the said method with other different nucleic acid molecules (polynucleotides) having nucleotide sequences non-overlapping with the former ones and having a size longer than 12 Kb. The selected nucleotide sequences (polynucleotides) used as probes are partly deleted of their natural frequently repeated sequences. The present invention concerns also improvements brought to the design of set of probe sequences for the detection of genetic rearrangements by hybridization as with fiber-FISH-like technologies such as Molecular Combing. The improvements described herein allow for high precision/high-resolution detection of rearrangements in time- and cost-efficient assays. This invention also relates to the use of probe sequences for diagnostics applications and companion diagnostics tests, to a method of detection of presence or absence of alterations in sequences and to a kit for the above uses. This is illustrated hereinafter with sets of nucleotide sequences corresponding to parts of at least two genes: MSH2 and MLH1 or to the regions of MSH2 and MLH1, whose mutations increase the risk of occurrence of human colorectal cancer.

The invention is related to the sets of polynucleotides or probes labeled or not which are specific of said genes. Presently, the detection of genetic rearrangements using current technologies is often insufficiently reliable for diagnostics use. Unlike most technologies used to detect genetic alterations, which suffer strong intrinsic limitations towards some types of rearrangements, direct technologies such as FISH or Fiber-FISH can intrinsically detect any type of rearrangements. Their use is mainly limited by their resolution. Molecular Combing, on the other hand, may reach sufficient resolution, but probe designs currently used fail to allow cost- and time-efficient high resolution analysis of rearrangements.

These improvements involve the combination within the same sets of probes of -typically shorter—probes designed to optimize the sensitive detection and precise measurement of rearrangements and—typically longer—probes to allow for fast and reliable detection of signals of interest when analyzing results. Alternative designs where the longer probes are replace with a combination of shorter probes having equivalent functions and effects are also disclosed.

Specific aspects of the invention based on the concept of combining small probes for resolution and long probes for ease of detection for the detection on one or more genomic region(s) of interest as disclosed in more detail below.

The invention thus concerns a method for detecting mutated or rearranged genomic polynucleotide (target) sequence comprising:

(a1) hybridizing a target genomic polynucleotide comprising one or more genomic region(s) of interest, where mutations or rearrangements are sought, to a set of short probes that bind to each region of interest without long gaps between the portions of the target sequence bound by the set of short probes, where on each genomic region a subset of short probes are selected so that when taken together they form a long contiguous stretch inside or outside the region of interest, and wherein the probes may optionally have frequent repetitive sequences removed and thus more generally are optionally devoid of such repetitive sequences; or

(a2) hybridizing a target genomic polynucleotide comprising one or more genomic region(s) of interest, where mutations or rearrangements are sought, to a set of short probes that bind to each region of interest without long gaps between the portions of the target sequence bound by the set of short probes and to one or more long (docking) probe(s) that bind to sequences near but outside of the region(s) of interest; wherein the sequence(s) of the long probe(s) does not overlap that of the short probes and wherein the short and/or long probes may optionally have frequent repetitive sequences removed and thus more generally are optionally devoid of such repetitive sequences;

(b) detecting the locations of hybridized probes on the genomic region(s) of interest; optionally,

(c) comparing the location of the hybridized probes on the target genomic polynucleotide sequence with one or more motifs based on the hybridization of said probes to a reference, control, normal, not mutated, or not rearranged genomic polynucleotide sequence; and optionally,

(d) correlating the presence of a mutated or rearranged genomic polynucleotide with a specific phenotype, disease, disorder, or condition.

The mutated or arranged genomic polynucleotide sequence can be obtained from a subject who has cancer or who is suspected to having cancer, for example, from a subject who has colorectal cancer or who is suspected of having colorectal cancer. In such a case, the short and long probes identify mutations or genomic rearrangements associated with colorectal cancer and a control or reference sample would not contain these mutations or rearrangements. The presence or risk of developing colorectal cancer is assessed by comparing a target genomic polynucleotide sequence with the reference and determining whether a mutation or rearrangement associated with colorectal cancer is present. This method can be practiced with specific probes corresponding to or derived from Probe sets 1, 2, 3 and 4. For colorectal cancer, a genomic region of interest can be selected from genes associated with this disease, such as MSH2, MLH1, MSH6, PMS2 or EPCAM.

Similarly, the method may be applied to samples obtained from subjects having or at risk of developing other kinds of cancer, such as breast cancer, ovary cancer, or lung cancer. The method may also be applied to samples obtained from subjects having or at risk of other kinds of diseases, disorders, or conditions, including cardiovascular disease, diabetes, neuromuscular disorders; such as myotonic dystrophy or spinal muscular atrophy or samples obtained from a subject who has, is suspected of having, or is suspected of being a carrier for a genetic or hereditary disease, disorder or condition, including known or unknown foetal genetic alterations. The sample can be obtained from a subject having a multigenic genetic or hereditary disease, disorder or condition or for a genetic or hereditary disease, disorder or condition associated with rearrangement of genomic DNA.

In some aspects of the invention, the sample will be obtained from a subject undergoing treatment for a disease, disorder or condition associated with a genomic or somatic genetic rearrangement and the results obtained are compared to results obtained at other time points before, during or after the termination of treatment. A companion test for evaluating the efficiency of a therapeutic drug on the mutated or rearranged nucleotide sequences of the gene or the region of the gene of interest can be performed using the short and long probes according to the invention.

Preferably, in the method described above, the hybridizing with the short and long probes in step a) will be performed simultaneously.

Preferably, the short probes range in length from 0.5 kb to 10 kb and the maximum size of the gaps between the short probes when they are bound to the target is 15 kb, preferably 12 kb and more preferably 10 kb.

The number of short probes employed in the method described above can range from 1, 2, 3 to 10, 15 or more.

The maximum size for the long probes is 150 kb and these probes preferably range from 12 kb to 40 kb in length. Preferably, in order to have “long probe(s) that bind to sequences near but outside of the region of interest”, distance between the long probes and the region of interest is no longer than 150 kb, and more preferably no longer than 75 kb and even more preferably no longer than 25 kb from the region of interest. The minimum size for a genomic region to be tested or targeted is 50 kb. The minimum number of regions of interest is one for a singleplex test and two or more for a multiplex test. Examples of combinations of short and/or long probes include at least one short (less than 10 kb) sequence and at least one non-overlapping long sequence (more than 15 kb), or at least one group of at least two short sequences, less than 10 kb each, which total group length is longer than 14 kb and less than 150 kb, hybridizing contiguously on the mutated or rearranged polynucleotide sequence. The short probes can comprise a set of contiguous probes that span a stretch of the genomic polynucleotide sequences inside or outside the region of interest that is at least 15 kb.

The long probes may have repetitive DNA sequences excluded. These repetitive sequences to be excluded would ordinarily appear more than once and more often than statistically predicted based on their length and base content, for example, repetitive sequences between 50 and 400 bp can be excluded, though shorter or longer repetitive sequences that decrease sensitivity or specificity of the method can be identified and excluded. An example of such a sequence is the repetitive Alu family DNA sequences.

According to an embodiment of the invention, in order for the probes, either short probes or long probes, to have repetitive sequences excluded, these probes are designed to hybridize in regions of the genome which are free of such repetitive sequences, i.e. which have less than 10% preferably less than 2% of the selected type(s) of repetititve sequences to be excluded.

In the method described above, the short and long probes are preferably fluorescently tagged and different components of the probe sets may be tagged with different labels, such as labels with different colors. Tagging provides one means to identify motifs or submotifs characteristic of a mutated or rearranged sequence.

Compositions or kits comprising a set of short probes or a combination of short and long probes as described herein and optionally one or more components for binding said probes to a polynucleotide, for performing molecular combing, and/or for detecting whether hybridization has occurred are also contemplated. For example, a composition containing the short and long probe(s) described above, wherein at least two of said probe sequences detect a genetic rearrangement by using Molecular Combing, said composition comprising either at least one short (<12 kb) sequence and at least one non-overlapping long sequence (>14 kb), or at least one group of at least two short sequences, less than 10 kb each, which total length is longer than 14 kb and less than 150 kb, hybridizing contiguously on the genetic target. The short probe(s) in such a composition may preferably range from 0.5 kb to 12 kb and the long probe(s) range from 14 kb to 40 kb. Frequent repetitive sequences described above may be removed from the probes. Examples of probe sequences are those that hybridize specifically on the MSH2 gene or in the region of the MSH2 gene or on the MLH1 gene or in the region of the MLH1 gene. Specific kinds of short probe sequence(s) where repetitive sequences have been removed include those selected from the group consisting of or comprising the sequences obtained by PCR amplification on human genomic DNA using the primer pairs described in Table 1 in the lines:

MSH2-v1

P3 (primer pairs P3a_MSH2-v1 to P3c_MSH2-v1, SEQ ID NO:21-26)

P4 (primer pairs P4a_MSH2-v1 to P4b_MSH2-v1, SEQ ID NO:27-30)

P5 (primer pairs P5a_MSH2-v1 to P5c_MSH2-v1, SEQ ID NO:31-36)P6 (primer pairs P6a_MSH2-v1 to P6b_MSH2-v1, SEQ ID NO:37-40)

P7 (primer pairs P7a_MSH2-v1 to P7c_MSH2-v1, SEQ ID NO:41-46)

P8 (primer pairs P8a_MSH2-v1 to P8b_MSH2-v1, SEQ ID NO:47-50)

P9 (primer pairs P9a_MSH2-v1 to P9c_MSH2-v1, SEQ ID NO:51-56)

P10 (primer pairs P10a_MSH2-v1 to P10b_MSH2-v1, SEQ ID NO:57-60) MLH1-v1

P3 (primer pairs P3a_MLH1-v1 to P3d_MLH1-v1, SEQ ID NO:95-102)

P4 (primer pairs P4a_MLH1-v1 to P4b_MLH1-v1, SEQ ID NO:103-106)

P5 (primer pairs P5a_MLH1-v1 to P5b_MLH1-v1, SEQ ID NO:107-110)

P6 (primer pair P6a_MLH1-v1, SEQ ID NO:111-112)

P7 (primer pair P7a_MLH1-v1, SEQ ID NO:113-114

P8 (primer pairs P8a_MLH1-v1 to P8d_MLH1-v1, SEQ ID NO:115-122)

and the short probes may be used in combination with the long probe sequence(s) selected from the group consisting of or comprising the sequences obtained by PCR amplification on human genomic DNA using the primer pairs described in Table 1 in the lines

MSH2-v1

P11 (primer pairs P11a_MSH2-v1 to P11c_MSH2-v1, SEQ ID NO:61-66)

P12 (primer pairs P12a_MSH2-v1 to P12e_MSH2-v1, SEQ ID NO:67-76)

MLH1-v1

P9 (primer pairs P9a_MLH1-v1 to P9c_MLH1-v1, SEQ ID NO:123-128)

P10 (primer pairs P10a_MLH1-v1 to P10e_MLH1-v1, SEQ ID NO:129-138).

Specific kinds of contiguous short probe sequence(s) forming long stretches include those selected from the group consisting of or comprising the sequences obtained by PCR amplification on human genomic DNA using the primer pairs described in Table 1 in the lines:

MSH2-v2

PE1-2 (primer pairs PE1_MSH2-v2 to PE2_MSH2-v2, SEQ ID NO:163-166) and

PE3-6 (primer pairs PE3_MSH2-v2 to PE5-6_MSH2-v2, SEQ ID NO:167-172), together forming one stretch;

PE9 (primer pairs E9_MSH2-v2 and I9-10_MSH2-v2, SEQ ID NO:185-188),

PE10 (primer pair E10_MSH2-v2, SEQ ID NO:189-190),

PE11 (primer pairs E11_MSH2-v2 and I11-12_MSH2-v2, SEQ ID NO:191-194),

PE12-14 (primer pairs E12_MSH2-v2 and E13-14_MSH2-v2, SEQ ID NO:195-198) and

PE15-16 (primer pairs E15_MSH2-v2 and E16_MSH2-v2, SEQ ID NO:199-202), together forming one stretch;

MLH1-v2

PE1-2 (primer pairs E1_MLH1-v2 and E2_MLH1-v2, SEQ ID NO:227-230),

PE3-4 (primer pairs I23_MLH1-v2, E3_MLH1-v2 and E4_MLH1-v2, SEQ ID NO:231-236),

PE5-6 (primer pairs E5_MLH1-v2 and E6_MLH1-v2, SEQ ID NO:237-240),

PE7-9 (primer pairs E7-8_MLH1-v2 and E9_MLH1-v2, SEQ ID NO:241-244) and

PE10-11 (primer pairs E10_MLH1-v2 and E11_MLH1-v2, SEQ ID NO:245-248), together forming one stretch;

The primers designed for the purpose of preparing short probes of the invention may have a sequence of 20 to 40 nucleotides and comprise in their 3′ end a sequence of at least 20 contiguous nucleotides that base pairs with the target. The primer sequence thus may also comprise additional nucleotides that do not base pair with the target in its 5′ end. The nucleotides which do not base pair may be useful for the construction of the primers or for the cloning of the amplified sequence resulting from polymerization starting from the primers. In a particular embodiment the sequence of the primer that hybridizes to the target is longer than 20 nucleotides. Molecular Combing is a powerful FISH-based technique for direct visualization of single DNA molecules that are attached, uniformly and irreversibly, to specially treated glass surfaces (Herrick and Bensimon, 2009); (Schurra and Bensimon, 2009). This technology considerably improves the structural and functional analysis of DNA across the genome and is capable of visualizing the entire genome at high resolution (in the kb range) in a single analysis. Another embodiment of the invention is a method for designing a set of short probes or set of short and long probes as described above comprising:

identifying a polynucleotide containing a genomic region of interest,

selecting long probe sequences outside of the genomic region of interest but within 100 kb of the closest probe in the region of interest, and preferably within 30 kb of the closest probe in the region of interest and optionally removing frequently repeated sequences from said long probe sequences,

selecting a short probe sequences from within the genomic region of interest so that no gaps longer than 20 kb, and preferably no gaps longer than 12 kb appear between the short probes; or selecting a series of short probes that together form a long continuous stretch that covers the genomic region of interest;

hybridizing the probes to a genomic polynucleotide comprising the genomic region of interest,

detecting the hybridized probes, and

determining which sets of probes form motifs that specifically identify the genomic sequence of interest from a reference genomic sequence.

The comparison of the location of the hybridized probes on the target genomic polynucleotide sequence with one or more motifs based on the hybridization of said probes to a reference, control, normal, not mutated, or not rearranged genomic polynucleotide sequence, as disclosed in the databanks or experimentally obtained on samples.

The techniques disclosed herein may be applied to diagnosis of disease as well as for the identification of genetic rearrangements associated with a disease, disorder or condition. They may also be used as companion diagnostics to study the responses of a subject or group of subjects who have particular rearrangements to therapy, responses to environmental agents, or the effects of lifestyle choices. Specifically, the diagnostic products and methods of the invention are useful for diagnosis and assessments for subjects having or at risk of developing colorectal cancer. High resolution barcodes allow multiplex analysis of patients for extended or expanded diagnosis at the levels of patient stratification/classification and prognosis. Thus, the techniques disclosed herein can also be used to predict the course and probably outcome of a disease, disorder or condition as well as the likelihood of progression, stability, or recovery. Multiplex high resolution barcodes also permit the identification of key genetic alterations in a subject that would benefit from a particular kind of therapy as well as a way to assess the reaction of a subject to a particular kind of therapy or therapeutic intervention. Specific embodiments of the invention include the following, which embodiments are especially carried out in vitro.

A method for detecting mutated or rearranged genomic polynucleotide sequence comprising: (a1) hybridizing a target genomic polynucleotide comprising one or more genomic region(s) of interest, where mutations or rearrangements are sought, to a set of short probes that bind to each region of interest without long gaps between the portions of the target sequence bound by the set of short probes said set of short probes optionally including or being in combination with a (sub)set of short probes selected so that on each genomic region some of the short probes when taken together form a long contiguous stretch inside or outside the region of interest and where the short probes may optionally have frequent repetitive sequences removed; or (a2) hybridizing a target genomic polynucleotide comprising one or more genomic region(s) of interest, where mutations or rearrangements are sought, to a set of short probes that bind to each region of interest without long gaps between the portions of the target sequence bound by the set of short probes and to one or more long (docking) probe(s) that bind to sequences near but outside of the region(s) of interest; wherein the sequence(s) of the long probe(s) does not overlap that of the short probes and wherein the short and/or long probes may optionally have some or all of the frequently repeating sequences removed; (b) detecting the locations of hybridized probes on the genomic region(s) of interest; optionally, (c) comparing the location of the hybridized probes on the target genomic polynucleotide sequence with one or more motifs based on the hybridization of said probes to a reference, control, normal, not mutated, or not rearranged genomic polynucleotide)sequence; and optionally, and/or (d) correlating the presence of a mutated or rearranged genomic polynucleotide with a specific phenotype, disease, disorder, or condition.

The invention relates in particular to the method herein described wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has cancer or who is suspected of having cancer or who is susceptible to have a genetic predisposition to cancer.

The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has colorectal cancer or who is suspected of having colorectal cancer or who is susceptible to have a genetic predisposition to colorectal cancer, wherein said short and long probes identify mutations or genomic rearrangements associated with colorectal cancer, wherein said control, not mutated or normal genomic sequence is obtained from a subject not at risk for colorectal cancer and wherein the detection of a genomic rearrangement; and assessing presence of or risk of developing colorectal cancer when said genomic rearrangement is detected. In this method the probes can hybridize specifically on the MSH2 gene, in the region of the MSH2 gene, on the MLH1 gene, or in the region of the MLH1 gene.

The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has breast cancer or who is suspected to having breast cancer or who is susceptible to have a genetic predisposition to breast cancer.

The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has ovarian cancer or who is suspected to having ovarian cancer or who is susceptible to have a genetic predisposition to ovarian cancer.

The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has lung cancer or who is suspected to having lung cancer or who is susceptible to have a genetic predisposition to lung cancer.

The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has a cardiovascular disease, disorder or condition or who is suspected of having cardiovascular disease, disorder or condition or who is susceptible to have a genetic predisposition to cardiovascular disease, disorder or condition.

The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has a diabetes or who is suspected of having diabetes or who is susceptible to have a genetic predisposition to diabetes.

The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has a neuromuscular disorder or who is suspected of having a neuromuscular disorder.

The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has, is suspected of having, or is susceptible of being a carrier for a genetic or hereditary disease, disorder or condition.

The invention also relates in a particular embodiment to a method wherein the short and long probe sequences are specific to human genes or to human genomic regions associated with cancer, colorectal cancer or a foetal genetic alteration known or unknown when said region or gene is mutated or genetically rearranged.

The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has, is suspected of having, or is suspected of being a carrier for a multigenic genetic or hereditary disease, disorder or condition or for a genetic or hereditary disease, disorder or condition associated with rearrangement of genomic DNA.

The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject undergoing treatment for a disease, disorder or condition associated with a genomic inherited or acquired rearrangement and the results obtained are compared to results obtained at other time points before, during or after the termination of treatment.

The invention relates to method of any of the embodiments described herein, characterized by the following features taken individually or in any combination: the hybridizing with the short and long probes in (a2) is performed simultaneously; the short probes are 10 kb or less; and/or the short probe(s) comprise at least one short (less than 10 kb) sequence and at least one non-overlapping long sequence (more than 12 kb), or at least one group of at least two short sequences, less than 5, 6, 7, 8, 9 or 10 kb each, total group length is longer than 12 kb and less than 150 kb, hybridizing contiguously on the mutated or rearranged polynucleotide sequence. In these methods the short probes may comprise a set of contiguous probes that span a stretch of the genomic polynucleotide sequences inside or outside the region of interest that is at least 14 kb; and/or the long probe(s) may comprise one or more docking probes of more than 14 kb and less than 40 kb. The long probe(s) may have a length of at least 14 kb and bind to a polynucleotide sequence outside the region of interest.

Both the long and short probes may be designed to exclude frequently occurring repetitive DNA sequences. These repetitive DNA sequences, which may be excluded from the long and short probes, will generally appear more than once and more often than statistically predicted based on their length and base content. For example, a repetitive DNA sequence between 50 and 400 contiguous nucleotides in length, which appear more than once and more often than statistically predicted based on their length and base content, can be excluded from the short and/or long probe(s). One example of a repetitive sequence that can be excluded from the short and long probes is or are members of the repetitive Alu family DNA sequences.

In some embodiments of the invention the probes in (b) of the first embodiment are fluorescently tagged so that they can be detected fluorometrically. In other embodiments in b) each probe is tagged with one of two or more fluorescent tags.

According to other embodiments of the methods above, motifs or easily identifiable subsets of the probes are detected and compared instead of every probe sequence.

The methods described above may employ at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more short probes. These short probes may each have a length of least 500, 600, 700, 800, 900 or more base pairs (bp). In some embodiments of the methods above, the probes will be selected so that the gaps between short probes in the genomic region of interest are no more than 12 kb each. In further embodiments the short probes will bind to a single contiguous genomic region of interest or the short probes can be selected to bind to more than one non-contiguous genomic region of interest. The long probes used in the method above may be selected so as to be no more than 20, 30 or 40 kb. The or each of the genomic region(s) of interest in the methods described above can be selected to be longer than 50 kb.

Another embodiment of the invention is a kit comprising a set of short probes or a set of short and a set of long probe(s); and optionally one or more components for binding said probes to a polynucleotide, for performing molecular combing, and/or for detecting whether hybridization has occurred; (i) wherein the short probes comprise a set of probes that taken together bind to a long continuous stretch of the genomic region of interest; or (ii) wherein the long probes bind to sequences outside the genomic region of interest, do not overlap the short probe sequences; and optionally, where the repetitive sequences have been removed from the long and/or short probes. A kit of the invention is suitable and/or is specific for use in a method of the invention as disclosed herein. In a particular embodiment its short and/or long probes are characterized by the features described herein in relation with the methods. Such a kit may be employed for or contain instructions for the detection of genomic rearrangements associated with colorectal cancer or genetic predisposition to colorectal cancer; for the detection of genomic rearrangements associated with breast cancer or genetic predisposition to breast cancer; for the detection of genomic rearrangements associated with ovarian cancer or genetic predisposition to ovarian cancer; for the detection of genomic rearrangements associated with lung cancer or genetic predisposition to lung cancer.

Another embodiment of the invention is a composition containing the short, or short and long probe(s) described by the first embodiment above, wherein at least two of said probe sequences detect a genetic rearrangement by using Molecular Combing, said composition comprising either (a) at least one short (less than 10 kb) sequence and at least one non-overlapping long sequence (more than 14 kb), or (b) at least one group of at least two short sequences, less than 10 kb each, which total length is longer than 14 kb and less than 150 kb, hybridizing contiguously on the genetic target. In this composition the short probe(s) can range from 0.5 kb to 9 kb and the long probe(s) can range from 14 kb to 40 kb. The size of the short probes may range from 0.5 to 9 kb and at least 90% of the frequent repetitive sequences can be been removed from the short probe sequences. This composition may contain probes sequences that hybridize specifically on the MSH2 gene or in the region of the MSH2 gene or on the MLH1 gene or in the region of the MLH1 gene.

In yet another embodiment the invention involves a method for designing short and long probes described herein in relation to methods comprising (a) identifying a polynucleotide containing a genomic region of interest, (b) selecting long probe sequences outside of the genomic region of interest but within 100 kb of the closest probe within the region of interest and optionally removing frequently repeated sequences from the long probe sequences, (c) selecting a set of short probe sequences from within the genomic region of interest so that no gaps longer than 15 kb appear between the short probes; or selecting a series of short probes that together form a long continuous stretch that covers the genomic region of interest; (d) hybridizing the probes to a genomic polynucleotide comprising the genomic region of interest, (e) detecting the hybridized probes, and (f) determining which sets of probes form motifs that distinguish the genomic sequence of interest from a reference genomic sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, which includes sub-parts identified as FIG. 1A, FIG. 1B, and FIG. 1C. (A) FIG. 1A: Dot-plot of MSH2 gene sequence on RP11-1084A21 BAC clone. (B) FIG. 1B: probe code v1 (without repetitive element) on RP11-1084A21. (C) FIG. 1C: probe code-v2 on RP11-1084A21. Diagonal lines are perfectly matched region of DNA between two sequences. Dots are representatives of repetitive elements. Higher density of dots (or grey band) are higher density of repetitive element.

FIG. 2, which includes sub-parts identified as FIG. 2A, FIG. 2B, and FIG. 2C. Dot plot analysis of MLH1 region. (A) FIG. 2A: Dot-plot of MLH1 gene sequence on RP11-426N19 BAC clone. (B) FIG. 2B: probe code v1 (without repetitive element) on RP11-426N19. (C) FIG. 2C: probe code-v2 on RP11-426N19.

FIG. 3, which includes sub-parts identified as FIG. 3A and FIG. 2B. Designed probe set for MSH2 by exclusion of repetitive element. A) FIG. 3A: theoretical probe set (labeled in red and green in microscopy experiments represented here in grey and black, respectively), and position of exon (small numbered dots). (B) FIG. 3B: actual hybridization image corresponding to MSH2-v1 probe set. Original microscopy images consist of three channel images where each channel is the signal from a given fluorophore—these are acquired separately in the microscopy procedure. These channels are represented here as different shades on a grayscale: green probes are shown in black and red probes in gray, while the background (absence of signal) is white. The aspect ratio was not preserved, signals have been “widened” (i.e. stretched perpendicularly to the direction of the DNA fiber) in order to improve the visibility of the probes.

FIG. 4, which includes sub-parts identified as FIG. 4A and FIG. 4B. Designed probe set for MLH1 by exclusion of repetitive element. A) FIG. 4A: theoretical probe set (red and green), and position of exon (purple dot). (B) FIG. 4B: actual hybridization image corresponding to MLH1-v1 probe set. The same color conventions are used for diagrams and microscopy images as in panels A and B of FIG. 3.

FIG. 5, which includes sub-parts identified as FIG. 5A and FIG. 5B. Designed probe set for MSH2 with docking probes (v2). (A) FIG. 5A: theoretical probe set). B) FIG. 5B: actual hybridization image corresponding to MSH2-v2 probe set. The color conventions in this and the other 3-color microscopy images (and corresponding diagrams) is as follows: blue probes are represented in black, green probes in dark gray, red probes in light gray and the background is white.

FIG. 6, which includes sub-parts identified as FIG. 6A and FIG. 6B. Designed probe set for with docking probes (v2). (A) FIG. 6A: theoretical probe set). (B) FIG. 6B: actual hybridization image corresponding to MLH1-v1 probe set. The same color conventions are used for diagrams and microscopy images as in FIG. 5.

FIG. 7, which includes sub-parts identified as FIG. 7A, FIG. 7B, and FIG. 2C. Validation of genomic rearrangement in MSH2 in LoVo cell line with v2 probe set. Sketches of both theoretical probe set (top; FIG. 7A) and validated rearrangement (middle, FIG. 7B) by molecular combing. The photo (bottom, FIG. 7C) is the recurrent abnormal signal set which corresponding to deletion from exon 3 to exon 8 of MSH2 (as in middle). The same color conventions are used for diagrams and microscopy images as in FIG. 5

FIG. 8, which includes sub-parts identified as FIG. 8A, FIG. 8B, and FIG. 8C. Validation of genomic rearrangement in MLH1 in SK-OV-3 cell line with v2 probe set. Sketches of both theoretical probe set (top; FIG. 8A) and validated rearrangement (middle; FIG. 8B) by molecular combing. The photo (bottom; FIG. 8C) is the representative (but few cases) signal set corresponding to the upper stream of MLH1 probe set (left side of theoretical probe set). The difference of observation number between MSH2 probe signal (normal) and MLH1 (a part of left side) clearly demonstrates that deletion of exon 4 to 19 in MLH1 is homozygous, (consistent with reference 7). Molecular combing test also revealed that the breakpoint of deletion is larger than previously reported (downstream probes from exon 19 are all deleted). The same color conventions are used for diagrams and microscopy images as in FIG. 5

Table 1. describes primer sequences and coordinates on human genomic DNA used for hybridization fragment synthesis to design the probes of the invention. These primers or variant therefore obtained by adding nucleotides in the ends of the described sequences and having up to 40 nucleotides, are part of the invention.

Table 2. Analysis of sequence of probe sets and their covering region. These sequences and the sets of probes that are disclosed in particular, are part of the invention.

Sequence of each of probe sets or region was subjected to RepeatMasker test and some of representative values are shown in the table. Sum length: sum up of sequence of all probes in each set. For MLH1 and MSH2 regions, this is the total length of each region. Repeat length: sum of sequences recognized as sorts of repeat in human genome. This includes sequences other than SINE. Total repeat. % of repeat length in sum length. SINE: % of sequences categorized as SINE in sum length. ALUs: % of sequences categorized as Alu family sequences in sum length.

DETAILED DESCRIPTION OF THE INVENTION

The above described strategies, for the reasons mentioned, are unsuitable to design a high-resolution code for diagnostics applications using technologies such as molecular combing.

In the present invention, the probes are defined as follows: a short probe is a nucleic acid sequence complementary to a genomic sequence, which probe can be detected with a given marker (such as a fluorochrome) once hybridized on the genomic sequence. One probe may be either made of (i) one single fragment covering the whole sequence, or of (ii) several exactly contiguous fragments, and/or (iii) slightly overlapping fragments (with an overlap less than 250 bp) and/or (iv) fragments separated by a very short gap (less than 1000 bp). With such short overlaps or gaps, using Molecular combing in our current setup, the fragments appears almost contiguous. The distance may be adjusted depending on the specific technique and experimental conditions. For example, with less resolutive conditions, longer gaps (less than 2 kb) or overlaps may be tolerated, provided fragments separated by such a gap still appear contiguous. Under more resolutive conditions, gaps should be shorter (less than 200 bp) in order for the fragments to appear contiguous. Short probes range in size from 500 bp to 10 kb.

A long probe is a nucleic acid sequence complementary to a genomic sequence, which probe can be detected with a given marker (such as a fluorochrome) once hybridized on the genomic sequence. One probe may be either made of (i) one single fragment covering the whole sequence, or of (ii) several exactly contiguous fragments, and/or (iii) slightly overlapping fragments (with an overlap less than 250 bp) and/or (iv) fragments separated by a gap (less than 3.5 kb), provided that more than 70% of the target sequence stretch is covered by probes (i.e. provided the gaps represent less than 30% of the target sequence). With such overlaps or gaps, using Molecular combing in our current setup, the fragments are efficiently detected. The distance may be adjusted depending on the specific technique and experimental conditions. For example, with less resolutive conditions, longer gaps (less than 5 kb each, representing in total less than 50% of the sequence) or overlaps may be tolerated, provided fragments separated by such gaps are still detected efficiently. Also, under such conditions, longer probes should be used (more than 20 kb) to allow for efficient detection. Under more resolutive conditions, gaps should be shorter (less than 2 kb) in order for the fragments to be efficiently detected, and probes may still be efficiently detected with shorter size (more than 10 kb). Long probes range in size from 12 kb to 150 kb.

In the present invention, the size of probes reflects the length of the genomic sequence where the probe hybridizes, independently of the number of strands in the DNA molecules. Therefore, a probe may be described as 1 kb (1 kilobase=1000 bases) or, indifferently, as 1000 bp (base pairs): in both cases, the probe hybridizes over 1000 bases of one of the strands of the target DNA molecule (and, if the probe is double stranded, also on the 1000 complementary bases of the other strand of the target molecule).

In the present invention, a “barcode” designates a specific motif formed by a set of probes labeled with different markers, where the motif characteristics are the lengths of the probes in the set, the lengths of the gaps separating successive probes and the colors in which the probes are detected (or, more generally, the markers with which the probes are labeled).

If a high coverage barcode is to be designed for high resolution, probe and space lengths need to be roughly in the 0.5 kb to 10 kb range (see above). This makes it unpractical to design probes that completely exclude rearrangements, and yet are spaced closely enough for the code to allow high location precision. On the other hand, some non-specific hybridization (i.e. hybridization of [parts of] a probe on genomic regions that are not the designed target of that probe) of a probe is acceptable when using a code strategy for the reading of signals. Indeed, in applications such as Southern blot where the hybridization of a single probe is assessed or aCGH where hybridization of every probe is considered separately, the non-specific hybridization of probes on even a very limited number of regions may lead to completely unusable results. To a lesser extent, this is also the case with multiple-probe applications such as FISH, since the resolution of FISH is insufficient to distinguish genomic regions as far apart as several tens of megabases: a single non-specific hybridization would lead to unusable results if it were located close enough to the targeted region.

In molecular combing and other similar applications using a code strategy, the quantity of non-specifically hybridized probes is not in issue per se. If a probe (or fragments of a probe) hybridizes even multiple times outside the region of interest, it is unlikely it will recreate a motif sufficiently similar to the code to be confusing. Also, non-specific hybridization over short sequences (<<1 kb), even within the region of interest, would most likely not be detected, unless they are sufficiently clustered to generate a long (>1 kb) stretch of non-specific hybridization. For the above reasons, the inventors have developed an alternative approach for the design of probes when the main issue is the design of a (several) high resolution code(s) in a (several) given genomic region(s). The main step of this approach relies only on the knowledge of the sequence of the region(s) themselves. When designing such a code, the major issue is to avoid significant non-specific hybridization within the regions of interest(s). Non-specific hybridization becomes an issue only if several probes display non-specific hybridization on neighboring sequences outside the region of interest. In the latter case, there is a risk that the pattern of probes resembles the original code, or a rearranged version of it, and this would likely lead to false conclusions. Although the invention described herein does not allow excluding such occurrences, this is relatively easily done once the method described herein has been used to exclude other non-specific hybridizations (see below).

The basis for this approach is the detection and exclusion of sequences that are repetitive within the region(s) of interest. For this, only the corresponding sequence(s) (the target sequence(s)) have to be known. One easy way to detect such repeats is the search for local sequence alignments within the target sequence(s), which can be done with e.g. a dot-plot comparison of each target sequence with itself and the other target sequences. A dot-plot is a graph with the two (sets of) sequences that are being compared forming the two axis, while dots are printed at every point where the coordinates correspond to a local homology. For example, if nucleotide x from sequence A (horizontal axis) matches nucleotide y from sequence B (vertical axis), then a dot will appear at the point with (x; y) coordinates. Graphically, local alignments appear as diagonal lines. Some more elaborate tools inspired from dot-plots are available, that compare short sequences (“words”, typically a few nucleotides/tens of nucleotides long) rather than single nucleotides, and display dots in various shades of gray depending on the extent of homology, thus allowing a direct visual reading of relaxed homologies (non-specific hybridization may well appear with incomplete homology). The comparison may also be done directly on both strands for one of the sequences, so homologies appear for both sense and reverse complement orientations. An example of such a tool is “Dotter” (ref. 4).

With these tools, very frequent repetitive sequences, such as Alu sequences in the Human genome, appear quite clearly, as they have local homologies with numerous other sequences within the target regions. Therefore, stretches with a high frequency of these sequences appear as a gray band (horizontal or vertical depending on whether the stretch is located on the vertical or horizontal axis). The exact appearance of these stretches with dot-plot display tools will depend on settings, and possibly word size. Settings were selected such that sequence stretches longer than 200 bp with more than 80% homology appear clearly and can be located with a roughly 10 bp precision.

A sequence of 200 bp or more that contains more than 10 significant homologous sequences (less than 1, 2, 3, 4, 5, 10, 15 or 20% nucleotide mismatch or insertion/deletion) within the regions of interest is a frequent repetitive sequence, prone to generate significant non-specific hybridization. It is generally possible to design probes in such a way that they are void of these frequent repetitive sequences, thus increasing the specificity and the high resolution of the present technology compared to the published previous methods.

“Docking” Probes

Although, as shown above, shorter probes make for more precise localization of breakpoints and measurement of deleted or amplified sequences, they are, generally speaking, more difficult to detect with fiber-fish techniques and molecular combing, as they appear as shorter stretches of signal, i.e., they are both smaller and less easy to distinguish from noise (fluorescent spots either unrelated to probes or to hybridization of probes). This is particularly true when considering automatic (computer-based) detection of signals.

It is therefore desirable to include longer probes in the code (for example, more than 12 kb and less than 150 kb, preferably more than 14 kb and less than 40 kb, in particular for the detection of genetic rearrangements in the regions of MSH2 or MLH1 genes). These probes would appear as actual lines (rather than spots), readily distinguishable from noise and easily detectable due to their size. Once the signals of interest are detected, the detection of other probes located on the same DNA fiber is easier.

This is especially true using technologies such as Molecular Combing where the linearity of the fibers implies the other probes, if any, are located in the alignment of the first probe. Therefore, the invention provides that the inclusion of longer (>12 kb, preferably >14 kb) probes in the set of probes is a step towards easier detection of signals of interest. Not all probes in the set need to be that long: in a fast and “rough” detection step, the long probes are sought, which allows the localization of signals of interest. These probes are called “docking probes” as they allow to “land” on the regions of interest efficiently. In a second step, the shorter probes are sought in the neighborhood of the docking probes (and more specifically in the case of Molecular Combing or related technologies, in the alignment of these probes). Although when performed by a human operator these steps can hardly be formally executed consecutively, if an operator may limit his search to longer probes, he can browse through images more rapidly, which would only allow him to detect these probes and spend more time on images where a docking probe is seen in order to look for other shorter probes. As the longer docking probes would locally diminish the location precision and the resolution of the code, it is preferable for them not to be located in the region where rearrangements are sought. This is possible if the probes are located near, but not in, the region of interest, e.g. at either end of this region.

If it is desirable to only consider complete signals in the analysis of a given region (i.e. signals covering the entire contiguous region), these longer probes may also be used to assess the integrity of the region: if there is a probe located at each end and both probes are present, no breakage of the fiber has occurred during the DNA preparation or stretching step. In cases where several non contiguous regions are analyzed in a single test, obviously each region has to have its “docking” probes in order to be correctly detected.

Continuous Stretch of Short Probes

An alternative to the “docking probes” approach above is to design the set of probes in such a way that at least some groups of shorter probes form a continuous stretch of signal. This is possible if probe sequences are adjacent. In that case, several probes, although short enough (less than 10 kb) to provide for sufficient resolution, may well combine to form a long enough (more than 14 kb) signal for fast and reliable detection. Indeed, if the operator may combine color channels to view images, this stretch would still appear as a long line rather than a spot, allowing its distinction from background noise. This is possible by using either common optical setups such as tri-color filters in fluorescence microscopy, or by using common image viewing software. In the case of automatic detection, it is also possible to use combined color information and therefore to make use of the very characteristic aspect of a multicolor line relatively to background spot-like noise.

Measurements

The probe designs described above likely lead to a large number of probes to be measured in a test. The usual approach for probe measurement is to measure all of the probes constituting a signal, as well as the gaps separating them. In a test with a large number of probes, the amount of work required for analyzing results is increased. In order to balance this, the invention relates to a more efficient designed approach for signal measurement. This approach consists in the measurement of subgroups of probes constituting easily recognizable motifs. The subgroups are two or several consecutive probes and the gaps between them, and possibly gaps at either end, chosen in order for their total length to remain within reasonably precise measurement range (10-30 kb).

There is likely to be a systematic bias in the measurement of digitalized images of fluorescent segments. Indeed, at the extremity of such a fragment, the intensity of the signal decreases gradually when moving away from the center, to reach the level of the background. Depending on where the operator/the software sets the threshold for the determination of the actual end there may be a systematic over- or under-estimation of the lengths. This bias is compensated for if the measured motifs have a probe at one end and a gap at the other. Therefore, it is preferable to design motifs in this way.

If a motif is found to have an abnormal length (different from the expected theoretical length) in a given sample, it remains possible to measure the probes and gaps within this motif in order to further precise the location of the rearrangement. With this approach, it is possible to measure in a fast and efficient way all of the signals for initial screening, while keeping the location precision allowed by small probes. The somewhat lower precision on measurements due to the larger size of the subgroups compared to the probes is essentially compensated for by the higher number of signals that can be measured within the same operator time.

Application to HNPCC—Rationale

Colorectal cancer is the 4th most frequent form of cancer in human and around 5% of the cancer is considered as a hereditary form. The most frequent form of hereditary colorectal cancer is known as Lynch syndrome, or HNPCC (hereditary non-polyposis colorectal cancer). HNPCC increases a lifetime risk of cancer development in up to 80% (lifetime risk is around 7% in normal population US). HNPCC also increases other cancers (endometrial, ovarian, stomach).

Genetic aspect of HNPCC is known as a result of mutation in some of Mismatch Repair (MMR) genes such as MSH2, MLH1, MSH6, PMS2, etc. MSH2 and MLH1 mutation accounts for more than 80% of all mutation of MMR genes in HNPCC. Both point mutation and large rearrangements are reported in mutation of those genes, and especially high % of large mutation in MSH2 is observed because of high level of small repetitive element in its genetic sequence. Today the molecular diagnosis is done after studies of familial cancer history, tumor characterization by microsatellite instability test.

Normally mutation one alleles of one of MMR genes is sufficient for molecular diagnosis of HNPCC. All HNPCC individuals have both wild and mutated genes. Point mutation of targeted MMR genes can be detected by sequencing of genes and current sequencing test investigates only the sequence of exons. In case of large rearrangements such as deletion and amplification (loss and gain of genetic elements, respectively), sequencing does not detect them because altered sequences do not exist, and frequently primer binding regions for sequencing are deleted. As a result, sequence information comes from only wild allele and gives false negative. Indeed, MSH2 and MLH1 genes are higher percentage of repetitive elements of SINE in their genetic sequence. To address this large rearrangement, the test should detect presence of deletion or amplification in the MMR genes. One approach is cartography of MMR genes with designed probes of hybridization. Causal large rearrangement has a wide range from sub-kb to loss of total gene (up to 100 kb). A given cartography has to be sensitive to this wide dynamic range of mutation. To cope with it specific probe design was done for MSH2 and MLH1 loci.

The present invention is also related to the detection of known or unknown genomic rearrangements. It is also related to kits containing probes according to the invention, for the detection of known or unknown genomic rearrangements and the associated pathologies, or associated predispositions to pathologies such as cancers or cardiovascular diseases for example.

EXAMPLES Application to HNPCC—Materials and Method

Probe Design v1

Each probe (probe means continuous hybridization signal, can consist of multiple cloned DNA fragments, e.g., probe 1 of MSH2-v2 covers a 15 kb stretch and consists of five cloned DNA fragments of 3 kb. Since gap or overlap of each junction of these five fragments are smaller than resolution (<50 bp), they are considered and indeed look like continuous single probe of 15 kb) on a region of gene sequence itself has a length between 3-6 kb. In case of larger rearrangement than probe or gap size, obvious change of color pattern of designed probe will be observed. As well as large rearrangement in probe region, such rearrangement is also detectable in gap region, meaning any rearrangement larger than 1 kb at any position in the target genes are detectable. This is a uniqueness of cartography method with high resolution probe hybridization. Other techniques (MLPA, aCGH) can detect only such rearrangement involving probe sequence. For genes with high frequency of large rearrangement such as MSH2 and MLH1, presence of repetitive element in their genetic sequence limits a freedom of probe design for the other technology. Inclusion of repetitive element sequence in their probe design increases false detection a lot, their probe designing has to be free of repetitive element in principle.

Probe sequence was chosen by a dot plot analysis. BAC clone sequence of each gene (RP11-1084A21 (Ch2:47,574,044-47,785,729 for MSH2, RP11-426N19 (Ch3: 36,992,516-37,161,490) for MLH1 was self-plotted and all grey bands region were excluded from the target region of PCR primer design. PCR primer set was designed in the target regions by Primer3plus PCR primer design tool (ref 6). A list of the primers' sequence is shown in table 1A and B. Exclusion of Alu repeat was verified by both dot-plot analysis and RepeatMasker (http://www._repeatmasker.org). FIG. 1B and FIG. 2B show a lot less grey band on dot-plot of probe fragment sequence on BAC clone than dot-plot of gene (containing Alu repeat) on BAC clone. This indicates that sequence of designed probes does not include recurrent repetitive sequence in this target regions. RepeatMasker analysis (with default setting of web server) also clearly shows a dramatic reduction of % of Alu sequence in designed probe sequence (table 2).

Probe Design v2

To facilitate “recognition” of barcodes on hybridization images, an alternative design of probe set (called v2) was done as said in “Docking” probe section. Design process is same as v1 except no exclusion of repetitive elements based on dot-plot. For v2 probe design, each probe was designed to have more than 3 kb length, close to limit to be recognized as “line”, and all exon sequences are covered by a probe stretch (no exons fall in gaps). Docking probes were designed on both extremities of each gene with 15-20 kb length. For MSH2-v2 code, specific probes covering EPCAM gene (see rationale part) was also included between two docking probes. DNA sequence of designed code v2 was subjected to dot-plot analysis to make sure that there is no segmental repeats inside of designed region (FIGS. 1C and 2C).

Cloning of Probe Fragments and Labeling for Hybridization Probe

Each fragment of probes was amplified by PCR, then the fragment was ligated into plasmid vector (pNEB193, pCR2.1-TOPO, pCRXL-TOPO). The ligation product was transformed into E. coli competent cells and end-sequences of cloned fragment were verified. Purified plasmid DNA set of each gene was separated into two (v1) or three (v2) gropes according to colors corresponding to theoretical barcodes (FIG. 3A and FIG. 4A for v1, FIG. 5 and FIG. 6 for v2 probe sets). Each group of plasmid DNA was labeled by random priming method. Either whole plasmids containing probe fragments' sequence or PCR amplified probe fragments were used as a template for random priming. There are three haptens to be used for three color detection, biotin (Biot), digoxigenin (Dig) and Alexa Fluor 488 (A488). Biot-labeling was done by BioPrime DNA labeling system (Invitrogen) with manufacture's instruction. For Dig and A488 labeling, dNTP mixture in the kit was replaced with home-blend dNTP mixtures (either 0.1 mM Digoxigenin-11-dUTP (Roche applied science) for Dig labeling or 0.1 mM ChromaTide® Alexa Fluor® 488-7-OBEA-dCTP (Invitrogen) for A488 labeling, 0.1 mM unmodified equivalent (dTTP or dCTP) and 0.2 mM each of other three deoxynucleotides in final labeling reaction solution.).

Sample DNA Preparation

3 cell human cell lines were used for validation for large rearrangement detection in either MSH2 or MLH1. Cell line GM17939 was used as non-mutated sample. Cell line LoVo was used for MSH2 rearrangement validation, which is homozygous for deletion of exon 3-exon8 in MSH2. Another cell line SK-OV-3 was used for rearrangement validation of MLH1, which was reported as homozygous deletion of exon 4-exon 19 in MLH1. For each cell line, cell culture was prepared according to cell bank's instruction. Cultured cells were harvested (for LoVo and SK-OV-3 when 50-70% confluency) or collected by centrifuge (for GM17939 when between 300,000-400,000 cells/ml of medium. Cell pellet was resuspended in 1×PBS/Trypsin mixture to have 1,000,000 cells in 45 μl the cell suspension was mixed with an equal volume of 1.2%(w/v) NuSieve GTG agarose solution in 1×PBS (melted and equilibrated at 50° C. in advance). The cell/agarose mixture as poured into a well of gel plug mold, followed by gelification at 4° C. for 30 min. the gelified agarose plug was immersed in a mixture of 2 mg/ml of Proteinase K, 1%(w/v) of sarcosyl in 0.5M EDTA (pH8.0, 250 μl for each plug). The agarose plug was incubated at 50° C. overnight.

Next day the incubated plug was washed in 1×TE (10 mM Tris-HCl, 1 mM EDTA, pH8.0) 3 times for 1 hour each. The DNA plug can be stored in 0.5mEDTA at 4° C. The washed plug was stained in 100 μl of 33 μM YOYO-1 (Invitrogen) in TE40.2 (40 mM Tris-HCl, 2 mM EDTA pH8.0) for 1 hour in the dark. The stained plug was heated at 68° C. in 1 ml of combing buffer (0.5M MES pH5.5) for 20 min, then cooled at 42° C. 10 min prior to add 1.5 unit of beta agarase I (NEB). Beta agarase treatment was carried overnight at 42° C. in the dark.

The following day the treated DNA solution was poured into a combing reservoir and a level of the solution in the reservoir was adjusted with additional combing buffer.

Molecular Combing

The DNA solution was set on a Molecular Combing Machine (MCS, Genomic Vision). Molecular combing was performed on a silanized coverslips (Combicoverslips, Genomic Vision). The combed coverslips was fixed at 68° C. for 4 hours, then used for hybridization (or stored at −20° C. until use).

Hybridization and Detection of Probe

For one hybridization, 5 μl of each of labeled probe solutions (of both MSH2 and MLH1) was combined together and with 10 μg of sonicated herring or salmon sperm DNA and 10 μg of human Cot1-DNA (only for V2 probe sets), then purified by standard ethanol precipitation. The precipitate was resuspended with 20 μl of hybridization buffer (50% formamide, 2×SSC, 1% SDS and BlockAid blocking solution (Invitrogen)). The resuspended probe solution was set on a clean glass slide and covered with a DNA combed coverslip. The slide was heated at 90° C. for 5 min for co-denaturation of both probe and combed DNA then incubated at 37° C. overnight with an humidity for hybridization between labeled probes and combed DNA.

The hybridized coverslips was washed in 50% Formamid/2×SSC solution 3 times for 5 min each, followed by another 3 times washing with 2×SSC for 5 min each. The washed coveslips was then developed with two or three layers of fluorescently labeled antibodies or streptavidin. For each layer, antibodies for all haptens were diluted 25 times in BlockAid blocking solution (20 μl in final volume) and incubated for 20 min at 37° C. For Biot, Streptavidin Alexa Fluor 594 (Invitrogen) was used for the 1^(st) and the 3^(rd) layer, biotin conjugated-goat anti-streptavidin antibody was used for the 2^(nd) layer. Fr Dig, mouse anti-Digoxin AMCA conjugated (Jackson immunoresearch) was for the 1^(st) layer, rat anti-mouse AMCA conjugated (Jackson immunoresearch) conjugated was for the 2^(nd), the goat anti-rat Alexa Fluore 350 conjugated (Invitrogen) was used for the 3^(rd) layer. For A488, rabbit anti-Alexa Fluor 488 (Invitrogen) was used for the 1^(st) layer, goat anti-rabbit Alexa Fluor 488 conjugated was used for the 2^(nd) layer (no third antibody for A488). After 20 min incubation of each layer of antibody, the coverslip was washed in 2×SSC/1% Tween 20 washing solution 3 times for 5 min each at room temperature. After the washing of 3^(rd) layer, the coverslip was rinsed in 1×PBS, followed by successive bath of 70, 90 and 100% ethanol for 1 min each. The coverslip was dried at room temperature prior to microscopy.

Signal Acquisition and Measurement

Fluorescent signal of developed antibody on the coverslip was obtained by standard epi-fluorescent microscope system or automated fluorescent microscope system (Image Xpress Micro, Molecular Devices) with custom scanning configuration for molecular combing signal. Every set of linearly aligned fluorescent signals and gaps was measured by ImageJ. Each measured set of signals (with color information) was subjected to pattern matching to determine position (if the set is a part of one of probe set) and orientation by comparison with the theoretical probe sets. All unclassified sets (did not match with any positions and orientations of theoretical probe sets) were subjected to similarity check between them to find whether recurrent abnormal pattern appears or not.

Application to HNPCC—Results

FIGS. 3B and 4B are representative images of signal from hybridized DNA. Some of probes look like “dot” rather than “line” as expected from their length. There are some “random” spots on images of hybridization, but these spots do not interfere recognition of designed code. Although signals of some small probes (arrowed in FIG. 3B, for example) is not evident to measure “length” of probe signals for size evaluation, measurement of “distance” between probe signals is possible and equivalent to measurement of the length of probe and gaps in normal probe set hybridization

FIGS. 5B and 6B are the representative image of hybridization signal of barcodes-v2. Fluorescent signals are more continuous than the signals of barcodes-v1, and easier to find docking probes and measure the length of each probe and gap. These barcodes-v2 were used to visualize large genomic rearrangements of characterized cancer cell lines, LoVo and SK-OV-3 (ref. 5).

FIG. 7 is a result of hybridization of barcodes v2 on combed DNA from LoVo cell line; LoVo cell line is homozygous for deletion in MSH2 (from exon 3 to 8). Hybridization slide had many normal (identical to theoretical code) signal of MLH1 gene but none of normal MSH2 signals. Instead, there was a recurrent signal of truncated form of the normal MSH2 signal (FIG. 7B). By deduction from the truncated signals, this truncation results from loss of probes and gaps corresponding to ex3 to 8 of MSH2 gene.

FIG. 8 is a result of barcodes-v2 on SK-OV-3 cell line DNA, homozygous for deletion in MLH1 (from ex4 to 19). Among many normal MSH2 signals, only a few signals of part of MLH1 (from probe 1 to probe 3) were observed. This means a lack of following sequence of MLH1, which is consistent with reference. Moreover, a lack of the right (downstream of MLH1) docking probe indicates that this deletion affects beyond exon 19 of MLH1.

The sequences selected to detect predisposition to colorectal cancer linked to rearrangements in the MSH2 genomic region or the MLH1 genomic region are preferably chosen among the following nucleotide sequences and their corresponding complementary sequences and are described as:

The short probes covering the MSH2 gene region and constituting contiguous stretches (PE1-2 and PE3-6 (SEQ ID NO:354-358); PE9 to PE15-16 (SEQ ID NO:365-373) in table 1 under the header MSH2-v2) and the other short probes covering MSH2 gene region (PE7 and PE8, SEQ ID NO:359-364 in table 1 under the header MSH2-v2); the long probes neighboring the MSH2 gene (tPP1, EPCAM5′, EPCAM3′ (SEQ ID NO:342-353) and cPP1 (SEQ ID NO:374-378) in table 1 under the header MSH2-v2); the short probes covering the MLH1 gene region and constituting a contiguous stretch (PE1-2 to PE10-11, SEQ ID NO:386-396, in table 1 under the header MLH1-v2) and the other short probes covering MLH1 gene region (PE12-13, PE14-15 and PE16-19, SEQ ID NO:397-401, in table 1 under the header MLH1-v2); the long probes neighboring the MLH1 gene (tPP1 (SEQ ID NO:379-385) and cPP1 (SEQ ID NO:402-408) in table 1 under the header MLH1-v2). For example, these probes may be obtained by amplification of the fragments using the primers listed in Table 1 under the headers MSH2-v2 (SEQ ID NO:139-212) and MLH1-v2 (SEQ ID NO:213-272).

INCORPORATION BY REFERENCE

Each document, patent, patent application or patent publication cited by or referred to in this disclosure is incorporated by reference in its entirety, especially with respect to the specific subject matter surrounding the citation of the reference in the text. However, no admission is made that any such reference constitutes background art and the right to challenge the accuracy and pertinence of the cited documents is reserved.

TABLE 1 Name SEQ ID SEQ ID of Name of NO NO probe fragment (fragment) For/Rev (primer) Sequence (5′-3′) start end MSH2-v1 P1 P1a_MSH2-v1 273 forward   1 TTCTTCCCAAGAGAGCCAAG 47595911 47595930 reverse   2 CTGTTTTGGAACCCCAAGTC 47597074 47597093 P1b_MSH2-v1 274 forward   3 GGCTTCAATCTGGGACTACG 47598716 47598735 reverse   4 GCTGTCACCGCCTCTTTTAC 47599478 47599497 P1c_MSH2-v1 275 forward   5 GCCAGGCACTTAGGCAGTAG 47600433 47600452 reverse   6 TTGGTCCTGACATCCTTTCC 47601671 47601690 P1d_MSH2-v1 276 forward   7 TTAGTTGAACAGGGCATGACAC  47602097 47602118 reverse   8 GGTAAAGGGGCCTGATGTC 47602743 47602761 P1e_MSH2-v1 277 forward   9 GAGCCTTGATGTTCCCTCTTAAC 47603695 47602743 reverse  10 ACCCAGATCCGAAACTGTTG  47604324 47603717 P1f_MSH2-v1 278 forward  11 CCGGCCTTACCTTTCATTTC 47605735 47604343 reverse  12 CCAGGATCCAGATCCAGTTG 47606965 47606984 P2 P2a_MSH2-v1 279 forward  13 GAGTTCCATGGCAGATCACC 47612521 47612540 reverse  14 GCAGCTTTCAATCACAAATCAG  47614067 47614088 P2b_MSH2-v1 280 forward  15 GAAGGGTTGGTCTTGCTGTC 47615115 47615134 reverse  16 ACCCTTTGCACCTCTCTGTG 47615632 47615651 P2c_MSH2-v1 281 forward  17 CCCGGTGTTGAATCATTTG 47616079 47616097 reverse  18 TTCAGCCCTGAAGGTAGAGG 47617513 47617532 P2d_MSH2-v1 282 forward  19 CTGGCCACTTTTTGGAAGAG 47618884 47618903 reverse  20 TGGGACGCAGAGTGATACAG 47619394 47619413 P3 P3a_MSH2-v1 283 forward  21 TTACTGGCGATCCTCAGAGC 47629651 47629670 reverse  22 AACGCCTCTTCCGTTGTATG 47631623 47631642 P3b_MSH2-v1 284 forward  23 GAAAGGACAGACCAAGTGCAG 47632605 47632625 reverse  24 AGCCTGTGCAGGGAAACTC 47633083 47633101 P3c_MSH2-v1 285 forward  25 AGTGGGATGCAGCTGAAAAG 47633591 47633610 reverse  26 CAACAGCATGGGAAAGATCC 47635238 47635257 P4 P4a_MSH2-v1 286 forward  27 TTGAAAGTTGGTCTTAGGAAGAGG 47643286 47643309 reverse  28 CCCAACAAACCTGGCTTTAG 47644179 47644198 P4b_MSH2-v1 287 forward  29 AGACGCCCAAAATCAACAAC 47645155 47645174 reverse  30 CCGCTTGCTGCTAAAAATTG 47646042 47646061 P5 P5a_MSH2-v1 288 forward  31 TGATTGCCAAGGAAGATTCAC 47657647 47657667 reverse  32 TGGAAGTAAATGCAGGTGCTC 47658763 47658783 P5b_MSH2-v1 289 forward  33 TCATTCTTGGGTGTTTCTCG 47659578 47659597 reverse  34 ATGGCGGTTTTGTGGAATAG 47660015 47660034 P5c_MSH2-v1 290 forward  35 GAGGGAGAGGGAACCTTTTG 47661699 47661718 reverse  36 GGGGACTATACCGCATTCAC 47662243 47662262 P6 P6a_MSH2-v1 291 forward  37 TGTTGATTCATGGGCATTTG 47669651 47669670 reverse  38 GCTGGGGAATCATGTATGAAG 47671879 47671899 P6b_MSH2-v1 292 forward  39 CATCAAGCACAGTTCCATTG 47672243 47672262 reverse  40 TTCTCTTTCCGTTTCCAGTG 47673113 47673132 P7 P7a_MSH2-v1 293 forward  41 GGAGCTTGGGAATTCAACTG 47678126 47678145 reverse  42 AGAAACGGGCATGTCATAGG 47679330 47679349 P7b_MSH2-v1 294 forward  43 CAGCCTACGTGCCCATTTC 47679649 47679667 reverse  44 TCAAAAGATGGCCAAAATGC 47681179 47681198 P7c_MSH2-v1 295 forward  45 GTGTTGCACCCATTAACTCG 47681915 47681934 reverse  46 AGCCTGGTGAGAGGTGACTG 47684723 47684742 P8 P8a_MSH2-v1 296 forward  47 CACGATGCCAGTCCAATTC 47689478 47689496 reverse  48 AAGGTGGACTTTAATGCAAAGG 47690835 47690856 P8b_MSH2-v1 297 forward  49 GGAGTGAGAGCGACACCTTG 47691634 47691653 reverse  50 CGACAGCTGACTGCTCTATGG 47694068 47694088 P9 P9a_MSH2-v1 298 forward  51 CACAATGGGAAAGGATGTAGC 47701939 47701959 reverse  52 CAGAGAAAAACACCCATGACC 47704112 47704132 P9b_MSH2-v1 299 forward  53 CACCGTGATCCTCCTTATTTC 47704395 47704415 reverse  54 GAACAAACAACGGATGAAAGG 47704945 47704965 P9c_MSH2-v1 300 forward  55 GTGGCATATCCTTCCCAATG 47705311 47705330 reverse  56 CCCCCAGACTGTGAATTAAGG 47705787 47705807 P10 P10a_MSH2-v1 301 forward  57 GATGCAGATCAGGGAAATGC 47711630 47711649 reverse  58 ATCTTGCTGGATGGACAAGG 47715272 47715291 P10b_MSH2-v1 302 forward  59 CTTAATCCTGAAAGGCAGGTG 47715788 47715808 reverse  60 TGTTTCTCAGGCAACCACAG 47717266 47717285 P11 P11a_MSH2-v1 303 forward  61 GAAACCACAGAATCGCCTTC 47731087 47731106 reverse  62 ACCTGGACAGTCCCACAGAC 47733482 47733501 P11b_MSH2-v1 304 forward  63 CAGTGCTTTTGCATCCTTCC 47734903 47734922 reverse  64 ATTTAATCCCCTGGCCAATC 47741649 47741668 P11c_MSH2-v1 305 forward  65 CACCTGTGCCCATCACATAG 47742239 47742258 reverse  66 GAGTCCCCTCTTGGAGAACC 47747829 47747848 P12 P12a_MSH2-v1 306 forward  67 AAAGCCATTTCCAGTGTCG 47753989 47754007 reverse  68 ATTGTGCAGCCAGAATTGAG 47758158 47758177 P12b_MSH2-v1 307 forward  69 TTCACAGCAAAGTGGCTCAG 47760593 47760612 reverse  70 GCTATTATGGGCTGCAAAGC 47764302 47764321 P12c_MSH2-v1 308 forward  71 TTCACTCCCAACAAGCACTG 47764863 47764882 reverse  72 TGCCCAGTCCTTTTTCACT 47765618 47765636 P12d_MSH2-v1 309 forward  73 AATCCCTCCTGCACACTTTC 47765925 47765944 reverse  74 AATGGATGCTTCCACTGTCC 47767687 47767706 P12e_MSH2-v1 310 forward  75 CCATCTGTGCAATTCCTTCC 47768105 47768124 reverse  76 GTTCAAAGGCAGAAGCCATC 47769886 47769905 MLH1-v1 P1 P1a_MLH1-v1 311 forward  77 GTCTGGATTCTTTCACAATGTAGC 37005551 37005576 reverse  78 TGCCAATCTTCTCCTCTGTTC 37006562 37006582 P1b_MLH1-v1 312 forward  79 AACCACCCAATGTGTTCACC 37006815 37006836 reverse  80 GTTCATTCCTGCGAGTAGGC 37007422 37007441 P1c_MLH1-v1 313 forward  81 GCCAAAGGTGGAAAATGTTG 37008987 37009008 reverse  82 GCCTTCTTCATGAAAGCACTG 37009873 37009893 P1d_MLH1-v1 314 forward  83 CCAGAAGGTGGAAGCTACAG 37011079 37011100 reverse  84 TGGGGTCAATGAAGCAAG 37011830 37011847 P1e_MLH1-v1 315 forward  85 ACATCGACCCAGAAAGTTCC 37012314 37012335 reverse  86 AATGTGCTTCGTACCACTGC 37012867 37012886 P1f_MLH1-v1 316 forward  87 AGCGTGCCATTGTACTCTCC 37013822 37013843 reverse  88 TTTCTGAGCCCATGATTTCC 37015267 37015286 P2 P2a_MLH1-v1 317 forward  89 GTGCCCAGCTAGTTCCATTC 37023623 37023644 reverse  90 TCAAGAGCGCTAATCCCATC 37025002 37025021 P2b_MLH1-v1 318 forward  91 TGCACATGCTCACTGAAAGAC 37026505 37026527 reverse  92 TTTTGCCTGCAAACTGACC 37027818 37027836 P2c_MLH1-v1 319 forward  93 CAGCAAGCACCAAATCACTG 37028305 37028326 reverse  94 AGTACCAGCCGTCCAAACTG 37032621 37032640 P3 P3a_MLH1-v1 320 forward  95 CCTGGCCAGAAAATTCATTG 37037607 37037628 reverse  96 ACCCTGCATTCCAAACTCAC 37039199 37039218 P3b_MLH1-v1 321 forward  97 GCAGTCCTTTGAGGATTTAGC 37042493 37042515 reverse  98 GAAAGATATCCAACAGGAAGTGAG 37043300 37043323 P3c_MLH1-v1 322 forward  99 TGGCCTTGTTTAAGGTCCTG 37043746 37043767 reverse 100 ATGGTCCTGCTGCTTCAGAG 37044723 37044742 P3d_MLH1-v1 323 forward 101 ACCCCGTCATAGCACAGTTC 37045295 37045316 reverse 102 CAAAGGCCATTCATCAGTTTC 37046439 37046459 P4 P4a_MLH1-v1 324 forward 103 GTGGCGTGATATCCTTGATTC 37053034 37053056 reverse 104 CTCTGGAATGACTGCTGCTG 37054289 37054308 P4b_MLH1-v1 325 forward 105 TGTGCTAGATGCCTCACTGG 37055182 37055203 reverse 106 TTGCCAAGAAGCACAACAAG 37058326 37058345 P5 P5a1_MLH1-v1 326 forward 107 CGGAGGCTCTACTGTTGGAC 37062345 37062366 reverse 108 TGCTGTCCACTCTGGAACTG 37064753 37064772 P5b_MLH1-v1 327 forward 109 ACATCAGAAGCCCTGGTTTG 37064571 37064592 reverse 110 GCTGGGAGTTCAAGCATCTC 37067377 37067396 P6 P6a_MLH1-v1 328 forward 111 TCGGTCTCAGTCACCATTTG 37072097 37072118 reverse 112 AACGCACCTGGCTGAAATAC 37075920 37075939 P7 P7a_MLH1-v1 329 forward 113 TGAACCTGCAATATCTCAGAGG 37079607 37079630 reverse 114 CTTACCGATAACCTGAGAACACC 37083805 37083827 P8 P8a_MLH1-v1 330 forward 115 CCCAGCCCATATATTTTAAAGC 37088387 37088410 reverse 116 CCAGCCACTCTCTGGACTATC 37089049 37089069 P8b_MLH1-v1 331 forward 117 GACATGGAGAGCCGAATCC 37089669 37089689 reverse 118 CCATTAAAATCGGGTCTGAAAG 37091446 37091467 P8c_MLH1-v1 332 forward 119 TCCAGACCCAGTGCACATC 37091887 37091907 reverse 120 CATGGTCAGTGCCATCAGAG 37092412 37092431 P8d_MLH1-v1 333 forward 121 AGCCTCCCAAAGTTAAGTGC 37092788 37092809 reverse 122 CCCAGCTAAAACCAACACAC 37093346 37093365 P9 P9a_MLH1-v1 334 forward 123 TGCCCTCAGCTACTCACTCC 37103285 37103306 reverse 124 AGGGCTCAGCCTTTAGGAAC 37105620 37105639 P9b_MLH1-v1 335 forward 125 GCCAGACTCTCGTTCCATTC 37106390 37106411 reverse 126 ACTCCCCATTCAGTCCCTTC 37111053 37111072 P9c_MLH1-v1 336 forward 127 AGGCACAACGTCAGGTTTTC 37114109 37114130 reverse 128 TTGGAATTTGTCCTGGTGTG 37117519 37117538 P10 P10a_MLH1-v1 337 forward 129 CACCATTGCCAACACTTCTG 37132898 37132919 reverse 130 GCCATTGGTTTGAAGGTGAC 37134201 37134220 P10b_MLH1-v1 338 forward 131 CTTAGTCACCGCCTGTCCTC 37134738 37134759 reverse 132 TAGCTGCATGTGGCTAATCG 37136986 37137005 P10c_MLH1-v1 339 forward 133 TGTGGCTCGCATTACATTTC 37137579 37137600 reverse 134 CGCTGTCATTACCTGCTTTG 37139742 37139761 P10d_MLH1-v1 340 forward 135 TGACCTCCAAAATCATCCAG 37140449 37140470 reverse 136 TTCTGAGCTAGGAGGTGCTG 37141321 37141340 P10e_MLH1-v1 341 forward 137 CCAGATTTGTAAATCCCTGTTC 37142008 37142031 reverse 138 TGTGTGGTTCTTAAGCATTCC 37142420 37142440 MSH2-v2 tPP1 tPP1a_MSH2-v2 342 forward 139 CTCAGTCCATCAGCCTCCTC 47574824 47577784 reverse 140 TGCTGTGCCCTGAGATTAAG 47574823 47577783 tPP1b_MSH2-v2 343 forward 141 AACTTAATCTCAGGGCACAGC 47577763 47580677 reverse 142 TGCAGCTTCAGCCTCTTG 47577762 47580676 tPP1c_MSH2-v2 344 forward 143 GCGTGGTGTTTCGTACCAG 47580604 47583785 reverse 144 GCTACTGGCCAGAAATCTTCC 47580603 47583784 tPP1d_MSH2-v2 345 forward 145 GCCCAGCCCTACTAAGGAAG 47583750 47586723 reverse 146 CTGTGCTCCCCTGCTAGAAC 47583749 47586722 tPP1e_MSH2-v2 346 forward 147 GTCGTCCTCTTCGACCTAGC 47586769 47589967 reverse 148 CAGCGCCTATTCTACAGCAG 47586768 47589966 EPCAM5′ EPCa_MSH2-v2 347 forward 149 TTCTTCCCAAGAGAGCCAAG 47595912 47598965 reverse 150 CCACCTTTAATCTGCCCAAC 47595911 47598964 EPCb_MSH2-v2 348 forward 151 GTGTTGGGCAGATTAAAGGTG 47598944 47602122 reverse 152 GCAGTGTCATGCCCTGTTC 47598943 47602121 EPCc_MSH2-v2 349 forward 153 CTCTTTGTGCCCTTTCTTTTG 47601745 47604931 reverse 154 AGTTCCTTAAAGCAGAGAAGATGG 47601744 47604930 EPCAM3′ EPCd_MSH2-v2 350 forward 155 AACCTGTCCCTGTGGATGAG 47604796 47607923 reverse 156 CCGAAGCATCCTTACATTCC 47604795 47607922 EPCe_MSH2-v2 351 forward 157 AATACCTGAACCCCCAAACC 47607722 47609876 reverse 158 CTCAGGCTATTTTCCAGATTCAC 47607721 47609875 EPCf_MSH2-v2 352 forward 159 GCATGCCTGTCATTCTGG 47609695 47612812 reverse 160 TCCAAGGGACTGAAACACAC 47609694 47612811 EPCg_MSH2-v2 353 forward 161 TTAGTGTGTTTCAGTCCCTTGG 47612790 47615135 reverse 162 GACAGCAAGACCAACCCTTC 47612789 47615134 PE1-2 E1_MSH2-v2 354 forward 163 GCACATTACGAGCTCAGTGC 47629942 47633045 reverse 164 CTACCAGGAGAACAGCACAGG 47629941 47633044 E2_MSH2-v2 355 forward 165 TGGGTTAGCATTGTGTTAGGTG 47632899 47636029 reverse 166 CCACAGGTGTGTGCCAATAG 47632898 47636028 PE3-6 E3_MSH2-v2 356 forward 167 AAGTTGCAGTTTGGCTGGTC 47635845 47638929 reverse 168 TTATCTCCAGCGGTGCTTATG 47635844 47638928 E4_MSH2-v2 357 forward 169 TACCATAAGCACCGCTGGAG 47638906 47642053 reverse 170 ACTCCACCAAGCCCAGTCTC 47638905 47642052 E5-6_MSH2-v2 358 forward 171 TTTAGAGACTGGGCTTGGTG 47642030 47644205 reverse 172 CTCTTCCCCAACAAACCTG 47642029 47644204 PE7 I6-7_MSH2-v2 359 forward 173 CCCAGTTTCAAGCGATTAAG 47651443 47654570 reverse 174 AGGAAAAGCATGTTATCTCCAG 47651442 47654569 E7_MSH2-v2 360 forward 175 TTCCGTAGCAGTAGGCATCC 47654026 47657170 reverse 176 TCACCACCACCAACTTTATGAG 47654025 47657169 I7-8_MSH2-v2 361 forward 177 TCCCAGATCTTAACCGACTTG 47656956 47660035 reverse 178 ATGGCGGTTTTGTGGAATAG 47656955 47660034 PE8 E8_MSH2-v2 362 forward 179 CCCAAACAACAGCATTAGCC 47670887 47673915 reverse 180 ACATCAGCCTCGGGACAAG 47670886 47673914 I8-9a_MSH2-v2 363 forward 181 TGAGCCCGTTGAATATAGTGG 47673830 47675514 reverse 182 AGTTTTCCTAAACGGGATGATG  47673829 47675513 I8-9b_MSH2-v2 364 forward 183 ATGGGTGTGCACGTGTGTAG 47675368 47678365 reverse 184 GCCATGTGCAATTGTGAGTC 47675367 47678364 PE9 E9_MSH2-v2 365 forward 185 CCTTGCATAGTTTGCTTCTGG 47688375 47690450 reverse 186 ATCATACAAGGGCCTGTTGG 47688374 47690449 I9-10_MSH2-v2 366 forward 187 AAACAGAAATCGCCCAACAG 47690418 47692377 reverse 188 TAGAGACCCACCCAGAAACG 47690417 47692376 PE10 E10_MSH2-v2 367 forward 189 CAGTCCGATTTCGTTTCTGG 47692347 47695506 reverse 190 CACACCTAGATTTGGCAATGG 47692346 47695505 PE11 E11_MSH2-v2 368 forward 191 TTCCATTGCCAAATCTAGGTG 47695484 47698468 reverse 192 GGCCCTAGTGTTTCCTTTCC 47695483 47698467 I11-12_MSH2-v2 369 forward 193 AAGGAAACACTAGGGCCTACAAC 47698452 47700589 reverse 194 CCTGGCCTCAGTACACTTTTG 47698451 47700588 PE12-14 E12_MSH2-v2 370 forward 195 AGGGATTCTCCCCACTTAGC 47700228 47702718 reverse 196 ATTGGAGGACTGGCTCAAAG 47700227 47702718 E13-14_MSH2-v2 371 forward 197 GCTTACCTTTGAGCCAGTCC 47702694 47705819 reverse 198 ACATGTTCCTACCCCCAGAC 47702693 47705818 PE15-16 E15_MSH2-v2 372 forward 199 TTTCTGCATCAGTTGGTTGC 47706613 47709532 reverse 200 GCCAAGTTATTGCTGCTTCAG 47706612 47709531 E16_MSH2-v2 373 forward 201 AGCCCTGTGAGGTTGGTAAC 47709413 47712504 reverse 202 TCAACAACAGCTGGAACTGC 47709412 47712503 cPP1 cPP1a_MSH2-v2 374 forward 203 CCTCTCAGGTCAGGCTTCTG 47730898 47733882 reverse 204 GCTCCCGCTAGAGAAACTCC 47730897 47733881 cPP1b_MSH2-v2 375 forward 205 GAGCGAAGCACCTAAAGCAC 47733879 47736946 reverse 206 AATTGGAGGGGGTGGAGTAG 47733878 47736945 cPP1c_MSH2-v2 376 forward 207 TGTCACCCAGTCAGGTCATC 47736760 47739876 reverse 208 TTGGAAGGAATCCAACAAGG 47736759 47739875 cPP1d_MSH2-v2 377 forward 209 TTCCCAGAACTCCTTGTTGG 47739846 47742962 reverse 210 TGCAAACCCCTTCTTTTCAG 47739845 47742961 cPP1e_MSH2-v2 378 forward 211 ACCCCATGCAGAAGCAATAG 47743027 47746218 reverse 212 AAATCCTGAAGGTGGGTTCC 47743026 47746217 MLH1v2 tPP1 tPP1b_MLH1-v2 379 forward 213 AGTTTCAGCCATGTTGCAG 37005587 37005605 reverse 214 TTGGCAAAATTGTGACTGAG 37007511 37007530 tPP1c_MLH1-v2 380 forward 215 CAGTCACAATTTTGCCAAGG 37007513 37007532 reverse 216 AGTTCGTGGCATCTAACTATCG 37009688  37009709 tPP1d_MLH1-v2 381 forward 217 GGTCCATGTGCTCCAAAAAG 37009460 37009479 reverse 218 TCCAAAACTGGGAACAAACC 37012624 37012643 tPP1e_MLH1-v2 382 forward 219 TGGTTTGTTCCCAGTTTTGG 37012623 37012642 reverse 220 TAGTGCACCACAGCCTCAAG 37015706 37015725 tPP1f_MLH1-v2 383 forward 221 GGATCACTTGAGGCTGTGGT 37015700 37015719 reverse 222 TCCAACAACTGCTGTGAAGG 37018677 37018696 tPP1g_MLH1-v2 384 forward 223 CACCACTGACCTTCCCTTCC 37018492 37018511 reverse 224 GCACAGAAAGACAAATATCACATGC 37020534 37020558 tPP1h_MLH1-v2 385 forward 225 CTCTTCCTCGTCTCCTCCTG 37020430 37020449 reverse 226 CCAATTCAATGCAAAACCTG 37022464 37022483 PE1-2 E1_MLH1-v2 386 forward 227 CGAGCAGCTCTCTCTTCAGG 37034273 37034292 reverse 228 AGCCTATAAGCACAGACCAACTG 37037250 37037272 E2_MLH1-v2 387 forward 229 TTCTCTAGCAGTTGGTCTGTGC 37037242 37037263 reverse 230 ACCCTGCATTCCAAACTCAC 37039199 37039218 PE3-4 I23_MLH1-v2 388 forward 231 GTTCATTTTGGGGCATGTTC 37039148 37039167 reverse 232 CTGCAACCTCCTTTGAGACAG 37042218 37042238 E3_MLH1-v2 389 forward 233 TGTCTCAAAGGAGGTTGCAG 37042219 37042238 reverse 234 CCAAAATGAAACTGCCTTCC 37044367 37044386 E4_MLH1-v2 390 forward 235 AGTTCCCTGGGTCATTTTCC 37044393 37044412 reverse 236 TTGTGGGAAGGCAAACTAGC 37046381 37046400 PE5-6 E5_MLH1-v2 391 forward 237 CCTGTGCTAGTTTGCCTTCC 37046376 37046395 reverse 238 GGTGGTCACCGTGGTAAAAG 37049553 37049572 E6_MLH1-v2 392 forward 239 GACCACCATGTGATTTCCAAG 37049566 37049586 reverse 240 TTGGTTGGCGGTTATTTCTC 37052510 37052529 PE7-9 E7-8_MLH1-v2 393 forward 241 TAACCGCCAACCAAGAAAAG 37052516 37052535 reverse 242 TGTCTGGAGACCTTCCCAAG 37055360 37055379 E9_MLH1-v2 394 forward 243 TGTGCTAGATGCCTCACTGG 37055182 37055201 reverse 244 ACTTGCCTACATTGCCCATC 37058175 37058194 PE10-11 E10_MLH1-v2 395 forward 245 ATGGGCAATGTAGGCAAGTC 37058176 37058195 reverse 246 TCTGCAGCCATGAATAAGTCC 37061070 37061090 E11_MLH1-v2 396 forward 247 CAGAGCTGAGGCGATAAATTG 37060960 37060980 reverse 248 TGCTCCTCTCCAATCCATTC 37063973 37063992 PE12-13 E12_MLH1-v2 397 forward 249 ATACTTTCCCAGCCCAAACC 37066434 37066453 reverse 250 TGATGGGGAAATGAGAGGAG 37069438 37069457 E13_MLH1-v2 398 forward 251 AGTGGCCTTTGTCCATTGAG 37069405 37069424 reverse 252 GACAGAGGTGAGAGCCTAGGAG 37071540 37071561 PE14-15 E14-15_MLH1-v2 399 forward 253 AATGTGTTGGGGAAGTGGTC 37081262 37081281 reverse 254 TTTGGACCACGGCTTTAGAC 37084405 37084424 PE16-19  E16-18_MLH1-v2 400 forward 255 AAGCTGAGGTCACGGATTTG 37087522 37087541 reverse 256 GATGGGCAAGTTTCATCTCC 37090568 37090587 E19_MLH1-v2 401 forward 257 TGGGACGAAGAAAAGGAATG 37090401 37090420 reverse 258 CACCGTGCCTCAGCCTATAC 37093446 37093465 cPP1 cPP1a_MLH1-v2 402 forward 259 GGACTAACCCACCTCCCTTC 37103239 37103258 reverse 260 GCTATAGGCAGCCCAGAGTG 37106372 37106391 cPP2a_MLH1-v2 403 forward 261 GCCAGACTCTCGTTCCATTC 37106390 37106409 reverse 262 AGGATTTGCCGTATGGACTC 37109450 37109469 cPP3a_MLH1-v2 404 forward 263 TCGCCCAAAGTCACAGTAAG 37109303 37109322 reverse 264 GATCTGTAGGCCCAGGATTTC 37112356 37112376 cPP4a_MLH1-v2 405 forward 265 AGGGGTTTCTATGGCTGGTC 37112314 37112333 reverse 266 CCTCCCTCAAACCTCCTCTC 37114423 37114442 cPP5a_MLH1-v2 406 forward 267 TTCTCCTGCAGAGGAAGAGG 37114369 37114388 reverse 268 TTGGAATTTGTCCTGGTGTG 37117519 37117538 cPP6a_MLH1-v2 407 forward 269 AAAGCCAGGGAGTGAATGG 37117566 37117584 reverse 270 ATGTGCATCTCCCTGGTGAC 37120703 37120722 cPP7a_MLH1-v2 408 forward 271 TGTGGGGAAATCAAAACCTG 37120784 37120803 reverse 272 GGGTAGACTGTGCGTGTGTG 37123930 37123949

TABLE 2 MLH1-v2 MLH1-v1 MLH1 MSH2-V2 MSH2-V1 MSH2 probe probe region probe probe region sum length 86366 55582 121536 106534 73609 171394 bp repeat 44684 18525 64712 53243 22133 94584 bp length total repeat 51.74 33.33 53.25 49.98 30.07 55.19 % SINE 24.93 2.58 23.85 34.68 5.03 35.95 % ALUs 22.38 0.09 21.85 32.85 0.76 34.15 %

REFERENCES

-   1. “Gene copy number variation and common human disease”, Fanciulli,     et. al. Clinical Genetics, 2010 77, 201-213 -   2. “Dynamic molecular combing: stretching the whole human genome for     high-resolution studies” Michalet, et al., Science 1997 277,     1518-1523 and “Bar code screening on combed DNA for large     rearrangemens of the BRCA1 and BRCA2 gene in French breast cancer     families”, Gad, et. al., J. Medical Genetics, 2002, 39, 817-821 -   3. “Sequence-based design of single-copy genomic DNA probes for     fluorescence in situ hybridization” Rogan, et. al., Genome Res. 2001     11, 1086-94. -   4. “A dot-matrix program with dynamic threshold control suited for     genomic DNA and protein sequence analysis”. Erik L. L. Sonnhammer     and Richard Durbin. Gene 1995, 167:GC1-10 -   5. “Microsatellite instability, mismatch repair deficiency and     genetic defects in human cancer cel lines”, Boyer J. C., et al.     Cancer Research 1995 55, 6063-6070, -   6. “Primer3Plus, an enhanced web interface to Primer3”, Untergasser     A., et al. Nucleic Acids Research 2007 35, W71-W74 

1-48. (canceled)
 49. A kit comprising a set of short probes or a set of short and a set of long probe(s); and optionally one or more components for binding said probes to a polynucleotide, for performing molecular combing, and/or for detecting whether hybridization has occurred; wherein said short probes 10 kb or less and said long probes are 12 kb or more; and (i) wherein the short probes comprise a set of probes that taken together bind to a continuous stretch of more than 12 kb of the genomic region of interest; or (ii) wherein the long probes bind to sequences outside the genomic region of interest and do not overlap the short probe sequences; and optionally, where the repetitive sequences have been removed from the long and/or short probes.
 50. A kit according to claim 49 for the detection of genomic rearrangements associated with a condition selected from the group consisting of: colorectal cancer or genetic predisposition to colorectal cancer, breast cancer or genetic predisposition to breast cancer, ovarian cancer or genetic predisposition to ovarian cancer, and lung cancer or genetic predisposition to lung cancer.
 51. A composition containing a set of short, or short and long probe(s), wherein at least two of said probes detect a genetic rearrangement by using Molecular Combing, said short probes binding to at least one region of interest without gaps longer than 15 kb between the portions of the target sequence bound by the short probes in each region of interest and said composition comprising either at least one short probe of less than 10 kb and at least one non-overlapping long probe of more than 14 kb that binds to a sequence near but outside of the region(s) of interest; or at least one group of at least two short probes, less than 10 kb each, which total length is longer than 14 kb and less than 150 kb, hybridizing contiguously on the genetic target.
 52. The composition of claim 51, wherein the short probe(s) range from 0.5 kb to 9 kb.
 53. The composition according to claim 51, wherein the long probe(s) range from 14 kb to 40 kb.
 54. The composition according to claim 51, wherein the size of the short probes range from 0.5 to 9 kb and wherein at least 90% of the frequent repetitive sequences have been removed from the short probes.
 55. The composition of according to claim 51, wherein the probe sequences hybridize specifically on the MSH2 gene or in the region of the MSH2 gene or on the MLH1 gene or in the region of the MLH1 gene.
 56. The composition according to claim 51, wherein said short probe sequence(s) are selected from the group consisting of the group of short probes obtained by amplification using the primer pairs disclosed as SEQ ID NO: 21-60, SEQ ID NO:95-122; SEQ ID NO:163-172; SEQ ID NO:185-202 and SEQ ID NO:227-248 or the long probe sequence(s) are selected from the group consisting of the group of long probe obtained by amplification using the primer pairs disclosed as SEQ ID NO: 61-76 and SEQ ID NO:123-138.
 57. A kit according to claim 49 wherein the short probes are at least 500 bp each.
 58. A kit according to claim 57 wherein the long probes are 14 kb or more, and optionally wherein the long probes are shorter than 150 kb.
 59. A kit according to claim 58 for the detection of genomic rearrangements associated with a condition selected from the group consisting of: colorectal cancer or genetic predisposition to colorectal cancer, breast cancer or genetic predisposition to breast cancer, ovarian cancer or genetic predisposition to ovarian cancer, and lung cancer or genetic predisposition to lung cancer.
 60. A kit according to claim 59, wherein sequences of more than 200 bp, of which more than 10 copies with less than 20% mismatch are found within the regions of interest, have been removed from the short and/or long probes.
 61. A kit according to claim 58, wherein sequences of more than 200 bp, of which more than 10 copies with less than 20% mismatch are found within the regions of interest, have been removed from the short and/or long probes
 62. A kit according to claim 59, wherein sequences of more than 200 bp, of which more than 10 copies with less than 20% mismatch are found within the regions of interest, have been removed from the short and/or long probes
 63. A composition according to claim 51, wherein sequences of more than 200 bp, of which more than 10 copies with less than 20% mismatch are found within the regions of interest, have been removed from the short and/or long probes.
 64. A composition according to claim 52, wherein sequences of more than 200 bp, of which more than 10 copies with less than 20% mismatch are found within the regions of interest, have been removed from the short and/or long probes.
 65. A composition according to claim 53, wherein sequences of more than 200 bp, of which more than 10 copies with less than 20% mismatch are found within the regions of interest, have been removed from the short and/or long probes.
 66. A composition according to claim 52, wherein the sizes of long probes range from 14 kb to 150 kb, and wherein sequences of more than 200 bp, of which more than 10 copies with less than 20% mismatch are found within the regions of interest, have been removed from the short and/or long probes. 